Multivalent DLL3 antibodies

ABSTRACT

Novel modulators, including antibodies and derivatives thereof, and methods of using such modulators to treat proliferative disorders are provided.

CROSS REFERENCED APPLICATIONS

This application claims priority from U.S. Provisional Application No.61/603,173 filed on Feb. 24, 2012, and U.S. Provisional Application No.61/719,803 filed on Oct. 29, 2012 each of which is incorporated hereinby reference in its entirety.

SEQUENCE LISTING

The instant application contains a sequence listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 19, 2013, isnamed 112.00.0013-00304_SL.txt and is 381,637 bytes in size.

FIELD OF THE INVENTION

This application generally relates to novel compounds, compositions andmethods of their use in diagnosing, preventing, treating or amelioratingproliferative disorders and any expansion, recurrence, relapse ormetastasis thereof. In a broad aspect, the present invention relates tothe use of delta-like ligand 3 (DLL3) modulators, including anti-DLL3antibodies and fusion constructs, for the treatment, diagnosis orprophylaxis of neoplastic disorders. Selected embodiments of the presentinvention provide for the use of such DLL3 modulators, includingantibody drug conjugates, for the immunotherapeutic treatment ofmalignancies preferably comprising a reduction in tumor initiating cellfrequency.

BACKGROUND OF THE INVENTION

Stem and progenitor cell differentiation and cell proliferation arenormal ongoing processes that act in concert to support tissue growthduring organogenesis and cell replacement and repair of most tissuesduring the lifetime of all living organisms. In the normal course ofevents cellular differentiation and proliferation is controlled bynumerous factors and signals that are generally balanced to maintaincell fate decisions and tissue architecture. Thus, to a large extent itis this controlled microenvironment that regulates cell division andtissue maturation where signals are properly generated based on theneeds of the organism. In this regard cell proliferation anddifferentiation normally occurs only as necessary for the replacement ofdamaged or dying cells or for growth. Unfortunately, disruption of cellproliferation and/or differentiation can result from a myriad of factorsincluding, for example, the under- or overabundance of various signalingchemicals, the presence of altered microenvironments, genetic mutationsor some combination thereof. When normal cellular proliferation and/ordifferentiation is disturbed or somehow disrupted it can lead to variousdiseases or disorders including proliferative disorders such as cancer.

Conventional treatments for cancer include chemotherapy, radiotherapy,surgery, immunotherapy (e.g., biological response modifiers, vaccines ortargeted therapeutics) or combinations thereof. Unfortunately, certaincancers are non-responsive or minimally responsive to such treatments.For example, in some patients tumors exhibit gene mutations that renderthem non-responsive despite the general effectiveness of selectedtherapies. Moreover, depending on the type of cancer and what form ittakes some available treatments, such as surgery, may not be viablealternatives. Limitations inherent in current standard of caretherapeutics are particularly evident when attempting to treat patientswho have undergone previous treatments and have subsequently relapsed.In such cases the failed therapeutic regimens and resulting patientdeterioration may contribute to refractory tumors which often manifestthemselves as a relatively aggressive disease that ultimately proves tobe incurable. Although there have been great improvements in thediagnosis and treatment of cancer over the years, overall survival ratesfor many solid tumors have remained largely unchanged due to the failureof existing therapies to prevent relapse, tumor recurrence andmetastases. Thus, it remains a challenge to develop more targeted andpotent therapies for proliferative disorders.

SUMMARY OF THE INVENTION

These and other objectives are provided for by the present inventionwhich, in a broad sense, is directed to methods, compounds, compositionsand articles of manufacture that may be used in the treatment of DLL3associated disorders (e.g., proliferative disorders or neoplasticdisorders). To that end, the present invention provides novel Delta-likeligand 3 (or DLL3) modulators that effectively target tumor cells and/orcancer stem cells and may be used to treat patients suffering from awide variety of malignancies. As will be discussed in more detailherein, there are at least two naturally occurring DLL3 isoforms orvariants and the disclosed modulators may comprise or associateselectively with one isoform or the other or with both. Moreover, incertain embodiments the disclosed DLL3 modulators may further react withone or more DLL family members (e.g., DLL1 or DLL4) or, in otherembodiments, may be generated and selected for so as to exclusivelyassociate or react with one or more DLL3 isoforms. In any event themodulators may comprise any compound that recognizes, competes,agonizes, antagonizes, interacts, binds or associates with a DLL3genotypic or phenotypic determinant (or fragment thereof) and modulates,adjusts, alters, regulates, changes or modifies the impact of the DLL3protein on one or more physiological pathways and/or eliminates DLL3associated cells. Thus, in a broad sense the present invention isgenerally directed to isolated DLL3 modulators and uses thereof. Inpreferred embodiments the invention is more particularly directed toisolated DLL3 modulators comprising antibodies (i.e., antibodies thatimmunopreferentially bind, react with or associate with at least oneisoform of DLL3) that, in particularly preferred embodiments, areassociated or conjugated to one or more cytotoxic agents. Moreover, asdiscussed extensively below, such modulators may be used to providepharmaceutical compositions useful for the prophylaxis, diagnosis ortreatment of proliferative disorders including cancer.

In selected embodiments of the invention, DLL3 modulators may comprise aDLL3 polypeptide or fragments thereof, either in an isolated form orfused or associated with other moieties (e.g., Fc-DLL3, PEG-DLL3 or DLL3associated with a targeting moiety). In other selected embodiments DLL3modulators may comprise DLL3 antagonists which, for the purposes of theinstant application, shall be held to mean any construct or compoundthat recognizes, competes, interacts, binds or associates with DLL3 andneutralizes, eliminates, reduces, sensitizes, reprograms, inhibits orcontrols the growth of neoplastic cells including tumor initiatingcells. In preferred embodiments the DLL3 modulators of the instantinvention comprise anti-DLL3 antibodies, or fragments or derivativesthereof, that have unexpectedly been found to silence, neutralize,reduce, decrease, deplete, moderate, diminish, reprogram, eliminate, orotherwise inhibit the ability of tumor inhaling cells to propagate,maintain, expand, proliferate or otherwise facilitate the survival,recurrence, regeneration and/or metastasis of neoplastic cells. Inparticularly preferred embodiments the antibodies or immunoreactivefragments may be associated with, or conjugated to, one or moreanti-cancer agents (e.g., cytotoxic agent).

With regard to such modulators it will be appreciated that compatibleantibodies may take on any one of a number of forms including, forexample, polyclonal and monoclonal antibodies, chimeric, CDR grafted,humanized and human antibodies and immunoreactive fragments and/orvariants of each of the foregoing. Preferred embodiments will compriseantibodies that are relatively non-immunogenic such as humanized orfully human constructs. Of course, in view of the instant disclosurethose skilled in the art could readily identify one or morecomplementarity determining regions (CDRs) associated with heavy andlight chain variable regions of DLL3 antibody modulators and use thoseCDRs to engineer or fabricate chimeric, humanized or CDR graftedantibodies without undue experimentation. Accordingly, in certainpreferred embodiments the DLL3 modulator comprises an antibody thatincorporates one or more complementarity determining regions (CDRs) asdefined in FIGS. 11A and 11B and derived from the light (FIG. 11A) orheavy (FIG. 11B) contiguous chain murine variable regions (SEQ ID NOS:20-203) set forth therein. Such CDR grafted variable regions are alsoshown in FIG. 11 comprising SEQ ID NOS: 204-213. In preferredembodiments such antibodies will comprise monoclonal antibodies and, ineven more preferred embodiments, will comprise chimeric, CDR grafted orhumanized antibodies.

Exemplary nucleic acid sequences encoding each of the amino acidsequences set forth in FIGS. 11A and 11B are appended hereto in thesequence listing and comprise SEQ ID NOS: 220 to 413. In this respect itwill be appreciated that the invention further comprises nucleic acidmolecules (and associated constructs, vectors and host cells) encodingdisclosed antibody variable region amino acid sequences including thoseset forth in the attached sequence listing. More particularly inselected embodiments compatible DLL3 modulators may comprise an antibodyhaving a light chain variable region and a heavy chain variable regionwherein said light chain variable region comprises an amino acidsequence having at least 60% identity to an amino acid sequence selectedfrom the group consisting of amino acid sequences as set forth in SEQ IDNO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQNO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ IDNO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQID NO: 78 SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86,SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO:96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO:114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO:150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQID NO: 160, SEQ ID NO: 162 SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO:168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO:186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200 and SEQ ID NO: 202 andwherein said heavy chain variable region comprises an amino acidsequence having at least 60% identity to an amino acid sequence selectedfrom the group consisting of amino acid sequences as set forth in SEQ IDNO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ IDNO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ IDNO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105,SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ IDNO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123,SEQ ID NO: 125, SEQ ID NO; 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ IDNO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141,SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ IDNO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159,SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ IDNO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177,SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ IDNO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195,SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201 and SEQ ID NO: 203. Inother preferred embodiments the selected modulators will comprise heavyand light chain variable regions that comprise 65, 70, 75 or 80%identity to the aforementioned murine sequences. In still otherembodiments the modulators will comprise heavy and light chain variableregions that comprise 85, 90 or even 95% identity to the disclosedmurine sequences.

In other preferred embodiments the selected modulators will comprise oneor more CDRs obtained from any of the foregoing light and heavy chainvariable region amino acid sequences. Accordingly, selected embodimentsof the invention include a DLL3 modulator comprising one or more CDRsfrom any one of SEQ ID NOS; 20 to 203. In still other embodiments themodulators of the instant invention will comprise any antibody orimmunoreactive fragment thereof that competes for binding with any ofthe foregoing modulators.

Another aspect of the invention comprises modulators obtained or derivedfrom SC16.3, SC16.4, SC16.5, SC16.7, SC16.8, SC16.10, SC16.11, SC16.13,SC16.15, SC16.18, SC16.19, SC16.20, SC16.21, SC16.22, SC16.23, SC16.25,SC16.26, SC16.29, SC16.30, SC16.31, SC16.34, SC16.35, SC16.36, SC16.38,SC16.39, SC16.41, SC16.42, SC16.45, SC16.47, SC16.49, SC16.50, SC16.52,SC16.55, SC16.56, SC16.57, SC16.58, SC16.61, SC16.62, SC16.63, SC16.65,SC16.67, SC16.68, SC16.72, SC16.73, SC16.78, SC16.79, SC16.80, SC16.81,SC16.84, SC16.88, SC16.101, SC16.103, SC16.104, SC16.105, SC16.106,SC16.107, SC16.108, SC16.109, SC16.110, SC16.111, SC16.113, SC16.114,SC16.115, SC16.116, SC16.117, SC16.118, SC16.120, SC16.121, SC16.122,SC16.123, SC16.124, SC16.125, SC16.126, SC16.129, SC16.130, SC16.131,SC16.132, SC16.133, SC16.134, SC16.135, SC16.136, SC16.137, SC16.138,SC16.139, SC16.140, SC16.141, SC16.142, SC16.143, SC16.144, SC16.147,SC16.148, SC16.149 and SC16.150. In other embodiments the invention willcomprise a DLL3 modulator having one or more CDRs from any of theaforementioned modulators.

In yet other compatible embodiments the instant invention will comprisethe CDR grafted or humanized DLL3 modulators hSC16.13, hSC16.15,hSC16.25, hSC16.34 and hSC16.56. Still other embodiments are directed toa DLL3 modulator comprising a humanized antibody wherein said humanizedantibody comprises a light chain variable region and a heavy chainvariable region wherein said light chain variable region comprises anamino acid sequence having at least 60% identity to an amino acidsequence selected from the group consisting of amino acid sequences asset forth in SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO:210 and SEQ ID NO: 212 and wherein said heavy chain variable regioncomprises an amino acid sequence having at least 60% identity to anamino acid sequence selected from the group consisting of amino acidsequences as set forth in SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO:209, SEQ ID NO: 211 and SEQ ID NO: 213. Moreover, as describedimmediately above nucleic acid sequences encoding the humanized heavyand light chain variable regions are set forth in the attached sequencelisting as SEQ ID NOS: 404-413.

Besides the aforementioned aspects, other preferred embodiments of theinstant invention will comprise DLL3 modulators associated or conjugatedto one or more drugs to provide modulator conjugates that may beparticularly effective in treating proliferative disorders (alone or incombination with other pharmaceutically active agents). More generally,once the modulators of the invention have been fabricated and selectedthey may be linked with, fused to, conjugated to (e.g., covalently ornon-covalently) or otherwise associated with pharmaceutically active ordiagnostic moieties or biocompatible modifiers. As used herein the term“conjugate” or “modulator conjugate” or “antibody conjugate” will beused broadly and held to mean any biologically active or detectablemolecule or drug associated with the disclosed modulators regardless ofthe method of association. In this respect it will be understood thatsuch conjugates may, in addition to the disclosed modulators, comprisepeptides, polypeptides, proteins, prodrugs which are metabolized to anactive agent in vivo, polymers, nucleic acid molecules, small molecules,binding agents, mimetic agents, synthetic drugs, inorganic molecules,organic molecules and radioisotopes. Moreover, as indicated above theselected conjugate may be covalently or non-covalently associated with,or linked to, the modulator and exhibit various stoichiometric molarratios depending, at least in part, on the method used to effect theconjugation.

Particularly preferred aspects of the instant invention will compriseantibody modulator conjugates or antibody-drug conjugates that may beused for the diagnosis and/or treatment of proliferative disorders. Suchconjugates may be represented by the formula M-[L-D]n where M stands fora disclosed modulator or target binding moiety, L is an optional linkeror linker unit, D is a compatible drug or prodrug and n is an integerfrom about 1 to about 20. It will be appreciated that, unless otherwisedictated by context, the terms “antibody-drug conjugate” or “ADC” or theformula M-[L-D]n shall be held to encompass conjugates comprising boththerapeutic and diagnostic moieties. In such embodiments antibody-drugconjugate compounds will typically comprise anti-DLL3 as the modulatorunit (M), a therapeutic or diagnostic moiety (D), and optionally alinker (L) that joins the drug and the antigen binding agent. In apreferred embodiment, the antibody is a DLL3 mAb comprising at least oneCDR from the heavy and light chain variable regions as described above.

As previously indicated one aspect of the invention may comprise theunexpected therapeutic association of DLL3 polypeptides with cancer stemcells. Thus, in certain other embodiments the invention will comprise aDLL3 modulator that reduces the frequency of tumor initiating cells uponadministration to a subject. Preferably the reduction in frequency willbe determined using in vitro or in vivo limiting dilution analysis. Inparticularly preferred embodiments such analysis may be conducted usingin vivo limiting dilution analysis comprising transplant of live humantumor cells into immunocompromised mice (e.g., see Example 17 below).Alternatively, the limiting dilution analysis may be conducted using invitro limiting dilution analysis comprising limiting dilution depositionof live human tumor cells into in vitro colony supporting conditions. Ineither case, the analysis, calculation or quantification of thereduction in frequency will preferably comprise the use of Poissondistribution statistics to provide an accurate accounting. It will beappreciated that, while such quantification methods are preferred,other, less labor intensive methodologies such as flow cytometry orimmunohistochemistry may also be used to provide the desired values and,accordingly, are expressly contemplated as being within the scope of theinstant invention. In such cases the reduction in frequency may bedetermined using flow cytometric analysis or immunohistochemicaldetection of tumor cell surface markers known to enrich for tumorinitiating cells.

As such, another preferred embodiment of the instant invention comprisesa method of treating a DLL3 associated disorder comprising administeringa therapeutically effective amount of a DLL3 modulator to a subject inneed thereof whereby the frequency of tumor initiating cells is reduced.Preferably the DLL3 associated disorder comprises a neoplastic disorder.Again, the reduction in the tumor initiating cell frequency willpreferably be determined using in vitro or in vivo limiting dilutionanalysis.

In this regard it will be appreciated that the present invention isbased, at least in part, upon the discovery that DLL3 immunogens aretherapeutically associated with tumor perpetuating cells (i.e., cancerstem cells) that are involved in the etiology of various proliferativedisorders including neoplasia. More specifically, the instantapplication unexpectedly demonstrates that the administration of variousexemplary DLL3 modulators can mediate, reduce, deplete, inhibit oreliminate tumorigenic signaling by tumor initiating cells (i.e., reducethe frequency of tumor initiating cells). This reduced signaling,whether by depletion, neutralization, reduction, elimination,reprogramming or silencing of the tumor initiating cells or by modifyingtumor cell morphology (e.g., induced differentiation, niche disruption),in turn allows for the more effective treatment of DLL3 associateddisorders by inhibiting tumorigenesis, tumor maintenance, expansionand/or metastasis and recurrence.

Besides the aforementioned association with cancer stem cells, there isevidence that DLL3 isoforms may be implicated in the growth, recurrenceor metastatic potential of tumors comprising or exhibitingneuroendocrine features or determinants (genotypic or phenotypic). Forthe purposes of the instant invention such tumors will compriseneuroendocrine tumors and pseudo neuroendocrine tumors. Intervention inthe proliferation of such tumorigenic cells using the novel DLL3modulators described herein, may thereby ameliorate or treat a disorderby more than one mechanism (e.g., tumor initiating cell reduction anddisruption of oncogenic pathway signaling) to provide additive orsynergistic effects. Still other preferred embodiments may takeadvantage of the cellular internalization of cell surface DLL3 proteinto deliver a modulator mediated anti-cancer agent. In this regard itwill be appreciated that the present invention is not limited by anyparticular mechanism of action but rather encompasses the broad use ofthe disclosed modulators to treat DLL3 associated disorders (includingvarious neoplasia).

Thus, in other embodiments the present invention will comprise the useof the disclosed modulators to treat tumors comprising neuroendocrinefeatures in a subject in need thereof. Of course the same modulators maybe used for the prophylaxis, prognosis, diagnosis, theragnosis,inhibition or maintenance therapy of these same tumors.

Other facets of the instant invention exploit the ability of thedisclosed modulators to potentially disrupt oncogenic pathways (e.g.,Notch) while simultaneously silencing tumor initiating cells. Suchmulti-active DLL3 modulators (e.g., DLL3 antagonists) may prove to beparticularly effective when used in combination with standard of careanti-cancer agents or debulking agents. Accordingly preferredembodiments of the instant invention comprise using the disclosedmodulators as anti-metastatic agents for maintenance therapy followinginitial treatments. In addition, two or more DLL3 antagonists (e.g.antibodies that specifically bind to two discrete epitopes on DLL3) maybe used in combination in accordance with the present teachings.Moreover, as discussed in some detail below, the DLL3 modulators of thepresent invention may be used in a conjugated or unconjugated state and,optionally, as a sensitizing agent in combination with a variety ofchemical or biological anti-cancer agents.

Accordingly another preferred embodiment of the instant inventioncomprises a method of sensitizing a tumor in a subject for treatmentwith an anti-cancer agent comprising the step of administering a DLL3modulator to said subject. Other embodiments comprise a method ofreducing metastasis or tumor recurrence following treatment comprisingadministering a DLL3 modulator to a subject in need thereof. In aparticularly preferred aspect of the invention the DLL3 modulator willspecifically result in a reduction of tumor initiating cell frequency isas determined using in vitro or in vivo limiting dilution analysis.

More generally preferred embodiments of the invention comprise a methodof treating a DLL3 associated disorder in a subject in need thereofcomprising the step of administering a DLL3 modulator to the subject. Inparticularly preferred embodiments the DLL3 modulator will be associated(e.g., conjugated) with an anti-cancer agent. In yet other embodimentsthe DLL3 modulator will internalize following association or bindingwith DLL3 on or near the surface of the cell. Moreover the beneficialaspects of the instant invention, including any disruption of signalingpathways and collateral benefits, may be achieved whether the subjecttumor tissue exhibits elevated levels of DLL3 or reduced or depressedlevels of DLL3 as compared with normal adjacent tissue. Particularlypreferred embodiments will comprise the treatment of disordersexhibiting elevated levels of DLL3 on tumorigenic cells as compared tonormal tissue or non-tumorigenic cells.

In yet another aspect the present invention will comprise a method oftreating a subject suffering from neoplastic disorder comprising thestep of administering a therapeutically effective amount of at least oneinternalizing DLL3 modulator. Preferred embodiments will comprise theadministration of internalizing antibody modulators wherein themodulators are conjugated or associated with a cytotoxic agent.

Other embodiments are directed to a method of treating a subjectsuffering from a DLL3 associated disorder comprising the step ofadministering a therapeutically effective amount of at least onedepleting DLL3 modulator.

In yet another embodiment the present invention provides methods ofmaintenance therapy wherein the disclosed effectors or modulators areadministered over a period of time following an initial procedure (e.g.,chemotherapeutic, radiation or surgery) designed to remove at least aportion of the tumor mass. Such therapeutic maintenance regimens may beadministered over a period of weeks, a period of months or even a periodof years wherein the DLL3 modulators may act prophylactically to inhibitmetastasis and/or tumor recurrence. In yet other embodiments thedisclosed modulators may be administrated in concert with knowndebulking regimens to prevent or retard metastasis, tumor maintenance orrecurrence.

As previously alluded to the DLL3 modulators of the instant inventionmay be fabricated and/or selected to react with both isoform(s) of DLL3or a single isoform of the protein or, conversely, may comprise apan-DLL modulator that reacts or associates with at least one additionalDLL family member in addition to DLL3. More specifically, preferredmodulators such as antibodies may be venerated and selected so that theyreact with domains (or epitopes therein) that are exhibited by DLL3 onlyor with domains that are at least somewhat conserved across multiple orall DLL family members.

In yet other preferred embodiments the modulators will associate or bindto a specific epitope, portion, motif or domain of DLL3. As will bediscussed in some detail below, both DLL3 isoforms incorporate anidentical extracellular region (see FIG. 1F) comprising at least anN-terminal domain, a DSL (Delta/Serrate/lag-2) domain and six EGF-likedomains (i.e., EGF1-EGF6). Accordingly, in certain embodiments themodulators will bind or associate with the N-terminal domain of DLL3(i.e. amino acids 27-175 in the mature protein) while in other selectedembodiments the modulators will associate with the DSL domain (i.e.amino acids 176-215) or epitope therein. Other aspects of the instantinvention comprise modulators that associate or bind to a specificepitope located in a particular EGF-like domain of DLL3. In this regardthe particular modulator may associate or bind to an epitope located inEGF1 (amino acids 216-249), EGF2 (amino acids 274-310), EGF3 (aminoacids 312-351), EGF4 (amino acids 353-389), EGF5 (amino acids 391-427)or EGF6 (amino acids 429-465). Of course it will be appreciated thateach of the aforementioned domains may comprise more than one epitopeand/or more than one bin. In particularly preferred embodiments theinvention will comprise a modulator that binds, reacts or associateswith the DSL domain or an epitope therein. In other preferredembodiments the invention will comprise modulators that bind, react orassociate with a particular EGF-like domain or an epitope therein. Inyet other preferred embodiments the modulators will bind, react orassociate with the N-terminal domain or an epitope therein.

With regard to modulator or antibody “bins” it will be appreciated thatthe DLL3 antigen may be analyzed or mapped through competitive antibodybinding using art-recognized techniques to define specific bins locatedon or along the protein. While discussed in more detail herein and shownin Examples 9 and 10 below, two antibodies (one of which may be termed a“reference antibody,” “bin delineating antibody” or “delineatingantibody”) may be considered to be in the same bin if they compete witheach other for binding to the target antigen. In such cases the subjectantibody epitopes may be identical, substantially identical or closeenough (either in a linear sense where they are separated by a few aminoacids or conformationally) so that both antibodies are sterically orelectrostatically inhibited or precluded from binding to the antigen.Such defined bins may be generally associated with certain DLL3 domains(e.g. the reference antibody will bind with an epitope contained in aspecific domain) though the correlation is not always precise (e.g.,there may be more than one bin in a domain or the bin may be definedconformationally and comprise more than one domain). It will beappreciated that those skilled in the art can readily determine therelationship between the DLL3 domains and empirically determined bins.

With regard to the present invention competitive binding analysis usingart-recognized techniques (e.g., ELISA, surface plasmon resonance orbio-layer interferometry) defined at least nine distinct bins, each ofwhich was found to contain a number of antibody modulators. For thepurposes of the instant disclosure the nine bins were termed bin A tobin I. Thus, in selected embodiments the present invention will comprisea modulator residing in a bin selected from the group consisting of binA, bin B, bin C, bin D, bin E, bin F, bin G, bin H and bin I. In otherembodiments the present invention comprise a modulator residing in a bindefined by a reference antibody selected from the group consisting ofSC16.3, SC16.4, SC16.5, SC16.7, SC16.8, SC16.10, SC16.11, SC16.13,SC16.15, SC16.18, SC16.19, SC16.20, SC16.21, SC16.22, SC16.23, SC16.25,SC16.26, SC16.29, SC16.30, SC16.31, SC16.34, SC16.35, SC16.36, SC16.38,SC16.39, SC16.41, SC16.42, SC16.45, SC16.47, SC16.49, SC16.50, SC16.52,SC16.55, SC16.56, SC16.57, SC16.58, SC16.61, SC16.62, SC16.63, SC16.65,SC16.67, SC16.68, SC16.72, SC16.73, SC16.78, SC16.79, SC16.80, SC16.81,SC16.84, SC16.88, SC16.101, SC16.103, SC16.104, SC16.105, SC16.106,SC16.107, SC16.108, SC16.109, SC16.110, SC16.111, SC16.113, SC16.114,SC16.115, SC16.116, SC16.117, SC16.118, SC16.120, SC16.121, SC16.122,SC16.123, SC16.124, SC16.125, SC16.126, SC16.129, SC16.130, SC16.131,SC16.132, SC16.133, SC16.134, SC16.135, SC16.136, SC16.137, SC16,138,SC16.139, SC16.140, SC16.141, SC16.142, SC16.143, SC16.144, SC16.147,SC16.148, SC16.149 and SC16.150. In still other embodiments theinvention will comprise modulators from bin A, modulators from bin B,modulators from bin C, modulators from bin D, modulators from bin E,modulators from bin F, modulators from bin G, modulators from bin H ormodulators from bin I. Yet other preferred embodiments will comprise areference antibody modulator and any antibody that competes with thereference antibody.

The term “compete” or “competing antibody” when used in the context ofthe disclosed modulators means binding competition between antibodies asdetermined by an assay in which a reference antibody or immunologicallyfunctional fragment substantially prevents or inhibits (e.g., greaterthan 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%.) specificbinding of a test antibody to a common antigen. Compatible methods fordetermining such competition comprise art known techniques such as, forexample, bio-layer interferometry, surface plasmon resonance, flowcytometry, competitive ELISA, etc.

Besides the aforementioned modulators, in selected embodiments theinvention comprises a pan-DLL modulator that associates with DLL3 and atleast one other DLL family member. In other selected embodiments theinvention comprises a DLL3 modulator that immunospecifically associateswith one or more isoform of DLL3 but does not immunospecificallyassociate with any other DLL family member. In yet other embodiments thepresent invention comprises a method of treating a subject in needthereof comprising administering a therapeutically effective amount of apan-DLL modulator. Still other embodiments comprise a method of treatinga subject in need thereof comprising administering a therapeuticallyeffective amount of a DLL3 modulator that immunospecifically associateswith one or more isoforms of DLL3 but does not immunospecificallyassociate with any other DLL family member.

Beyond the therapeutic uses discussed above it will also be appreciatedthat the modulators of the instant invention may be used to detect,diagnose or classify DLL3 related disorders and, in particular,proliferative disorders. They may also be used in the prognosis and/ortheragnosis of such disorders. In some embodiments the modulator may beadministered to the subject and detected or monitored in vivo. Those ofskill in the art will appreciate that such modulators may be labeled orassociated with effectors, markers or reporters as disclosed below anddetected using any one of a number of standard techniques (e.g., MRI,CAT scan, PET scan, etc.).

Thus, in some embodiments the invention will comprise a method ofdiagnosing, detecting or monitoring a DLL3 associated disorder in vivoin a subject in need thereof comprising the step of administering a DLL3modulator.

In other instances the modulators may be used in an in vitro diagnosticsetting using art-recognized procedures (e.g., immunohistochemistry orIHC). As such, a preferred embodiment comprises a method of diagnosing ahyperproliferative disorder in a subject in need thereof comprising thesteps of:

-   -   a. obtaining a tissue sample from said subject;    -   b. contacting the tissue sample with at least one DLL3        modulator; and    -   c. detecting or quantifying the DLL3 modulator associated with        the sample.

Such methods may be easily discerned in conjunction with the instantapplication and may be readily performed using generally availablecommercial technology such as automatic plate readers, dedicatedreporter systems, etc. In selected embodiments the DLL3 modulator willbe associated with tumor perpetuating cells (i.e., cancer stem cells)present in the sample. In other preferred embodiments the detecting orquantifying step will comprise a reduction of tumor initiating cellfrequency which may be monitored as described herein.

In a similar vein the present invention also provides kits or devicesand associated methods that are useful in the diagnosis and monitoringof DLL3 associated disorders such as cancer. To this end the presentinvention preferably provides an article of manufacture useful fordetecting, diagnosing or treating DLL3 associated disorders comprising areceptacle containing a DLL3 modulator and instructional materials forusing said DLL3 modulator to treat, monitor or diagnose the DLL3associated disorder. In selected embodiments the devices and associatedmethods will comprise the step of contacting at least one circulatingtumor cell.

Other preferred embodiments of the invention also exploit the propertiesof the disclosed modulators as an instrument useful for identifying,characterizing, isolating, sectioning or enriching populations orsubpopulations of tumor initiating cells through methods such asimmunohistochemistry, flow cytometric analysis including fluorescenceactivated cell sorting (FACS) or laser mediated sectioning.

As such, another preferred embodiment of the instant invention isdirected to a method of identifying, isolating, sectioning or enrichinga population of tumor initiating cells comprising the step of contactingsaid tumor initiating cells with a DLL3 modulator.

The foregoing is a summary and thus contains, by necessity,simplifications, generalizations, and omissions of detail; consequently,those skilled in the art will appreciate that the summary isillustrative only and is not intended to be in any way limiting. Otheraspects, features, and advantages of the methods, compositions and/ordevices and/or other subject matter described herein will becomeapparent in the teachings set forth herein. The summary is provided tointroduce a selection of concepts in a simplified form that are furtherdescribed below in the Detailed Description. This summary is notintended to identify key features or essential features of the claimedsubject matter, nor is it intended to be used as an aid in determiningthe scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F are various representations of DLL3 including nucleic acidor amino acid sequences wherein full length mRNAs containing the ORFs(underlined) encoding DLL3 isoforms are depicted in FIGS. 1A and 1B (SEQID NOS: 1 and 2), FIGS. 1C and 1D provide the translation of the ORFsdenoted in FIGS. 1A and 1B (SEQ ID NOS: 3 and 4), respectively, withunderlined residues indicating the predicted transmembrane spanningdomain for each protein isoform, FIG. 1E depicts the alignment of thetwo protein isoforms to illustrate the sequence differences in thecytoplasmic termini of each isoform, again with the underlined residuesindicating the predicted transmembrane spanning domain and FIG. 1Fprovides a schematic representation of the extracellular region of DLL3protein illustrating the positions of the various domains;

FIGS. 2A and 2B are tabular representations of the percent identity atthe protein level between DLL3 and other Delta-like family members inthe human genome (FIG. 2A), or the closest human isoform of DLL3 andrhesus monkey, mouse and rat DLL3 proteins (FIG. 2B);

FIG. 3 schematically illustrates genetic interactions between several“master” genes relevant to cell fate choices leading to eitherneuroendocrine or non-neuroendocrine phenotypes (arrows indicatingpromotion of gene expression and barred arrows indicating inhibition ofgene expression), in which the expression of the transcription factorASCL1 both initiates a gene cascade (open arrow) leading to aneuroendocrine phenotype while simultaneously activating DLL3, which inturn suppresses NOTCH1 and its effector HES1, both of which are normallyresponsible for the suppression of ASCL1 and the activation of genecascades leading to a non-neuroendocrine phenotype;

FIGS. 4A and 4B are tabular (FIG. 4A) and graphical (FIG. 4B) depictionsof gene expression levels of DLL3 and, in FIG. 4A, other Notch pathwaygenes or genes associated with a neuroendocrine phenotype as measuredusing whole transcriptome (SOLiD) sequencing of RNA derived from tumorcell subpopulations or normal tissues;

FIG. 5 is a graphical depiction of the relative expression levels ofDLL3 mRNA transcript variants 1 and 2 as determined by wholetranscriptome (SOLiD) sequencing in selected non-traditional xenograft(NTX) tumors derived from lung cancers;

FIGS. 6A-6D show gene expression data and clustering of tumorsexhibiting neuroendocrine features wherein FIG. 6A depicts unsupervisedclustering of microarray profiles for 46 tumor lines and 2 normaltissues comprising selected tumors and normal control tissues, FIGS. 6Band 6C are tabular representations of normalized intensity valuescorresponding to relative expression levels of selected genes related toneuroendocrine phenotypes (FIG. 6B) or the Notch signaling pathway (FIG.6C) wherein unshaded cells and relatively low numbers indicate little tono expression and darker cells and relatively higher numbers indicatehigher expression levels and FIG. 6D is a graphical representationshowing relative expression levels of HES6 mRNA in various tumors andnormal tissues as measured using qRT-PCR;

FIG. 7 is a graphical representation showing relative expression levelsof DLL3 transcripts as measured by qRT-PCR in a variety of RNA samplesisolated from normal tissues, primary, unpassaged patient tumorspecimens (denoted with “p0”), or bulk NTX tumors derived from lung,kidney and ovarian neoplasia wherein specific NTX lung tumors aregrouped by small cell lung cancer (SCLC) and non-small cell lung cancer(NSCLC) (denoted with p1, p2, p3 or p4 to reflect the number of passagesthrough mice), wherein the tumor type is denoted using the abbreviationsset forth above;

FIGS. 8A-8C are graphical representations showing the relative (FIG. 8A)or absolute (FIG. 8B) gene expression levels of human DLL3 as measuredby qRT-PCR in whole tumor specimens (grey dot) or matched normaladjacent tissue (NAT; white dot) from patients with one of eighteendifferent solid tumor types while FIG. 8C shows the relative proteinexpression of human DLL3 as measured using an electrochemiluminescentsandwich ELISA assay;

FIG. 9 provides graphical representations of flow cytometry-baseddetermination of surface protein expression of various Notch receptorsand ligands (e.g., DLL1, DLL4) in individual human tumor cellpopulations derived from kidney, ovarian and small cell lung NTX tumors,displayed as histogram plots (black line) referenced to fluorescenceminus one (FMO) isotype-control stained population (solid gray) withindicated mean fluorescence intensities (MFI);

FIGS. 10A-10D provide, respectively, the cDNA sequence (FIG. 10A; SEQ IDNO: 5) and the amino acid sequence (FIG. 10B; SEQ ID NO: 6) encodingmature murine DLL3 protein cloned into a lentiviral expression vectorand the cDNA sequence (FIG. 10C; SEQ ID NO:7) and the amino acidsequence (FIG. 10D; SEQ ID NO: 8) encoding mature cynomolgus DLL3protein cloned into a lentiviral expression vector where the vectors areused to generate cells overexpressing murine and cynomolgus DLL3;

FIGS. 11A and 11B provide, in a tabular form, contiguous amino acidsequences (SEQ ID NOS: 20-213) of light and heavy chain variable regionsof a number of murine and humanized exemplary DLL3 modulators isolated,cloned and engineered as described in the Examples herein;

FIG. 12 sets forth biochemical and immunological properties of exemplaryDLL3 modulators as well as their ability to kill KDY66 NTX cell in vitroas represented in a tabular format;

FIGS. 13A-13C illustrate binding characteristics of selected modulatorswherein FIGS. 13A and 13B show comparative binding characteristics of aselected murine modulator and its humanized counterpart using surfaceplasmon resonance while FIG. 13C provides certain properties ofhumanized constructs in a tabular form;

FIGS. 14A and 14B depict, in schematic and graphical form respectively,the results of domain level mapping analysis of exemplary DLL3modulators isolated, cloned and engineered as described in the Examplesherein (FIG. 14A) and a correlation between the binding domain ofselected modulators and the ability to kill DLL3 expressing KDY66 NTXcells in vitro (FIG. 14B);

FIGS. 15A-15C are flow cytometry histograms showing DLL3 expressionusing the exemplary anti-DLL3 modulator SC16.56 on naive 293 cells (FIG.15A), 293 cells engineered to over-express human DLL3 proteins(h293-hDLL3; FIG. 15B) or 293 cells engineered to over-express murineDLL3 protein (h293-mDLL3; FIG. 15C);

FIGS. 16A-16F comprise flow cytometry histograms (FIGS. 16A-16C) andimmunohistochemistry results in a tabular form (FIGS. 16D-16F)illustrating, respectively, relatively high surface expression of DLL3using the exemplary anti-DLL3 modulator SC16.56 on live human cells fromovary (OV26; FIG. 16A), kidney (KDY66; FIG. 16B) and a lung large cellneuroendocrine carcinoma (LU37; FIG. 16C) NTX tumors and the expressionof DLL3 protein in various NTX tumors (FIG. 16D) and primary small cellcarcinoma (FIG. 16F) tumor cells while demonstrating that normal tissuelack DLL3 expression (FIG. 16E);

FIGS. 17A-17C illustrate the ability of the disclosed modulators toeffectively direct cytotoxic payloads to cells expressing DLL3 whereinFIG. 17A documents the ability of exemplary modulators to kill KDY66 NTXtumors or 293 cells overexpressing hDLL3, and FIGS. 17B and 17Cdemonstrate the ability of disclosed modulators to deliver cytotoxicpayloads to OV26 (FIG. 17B) and LU37 (FIG. 17C) where the downwardsloping curve is indicative of cell killing through internalizedcytotoxin;

FIGS. 18A-18E illustrate various properties of the disclosed modulatorswherein FIGS. 18A and 18C demonstrate by flow cytometry that DLL3 NSHPKDY66 and naïve KDY66 have expression of DLL3 while expression of DLL3was efficiently knocked down in DLL3HP2 KDY66 cells, FIG. 18B shows thatgrowth of DLL3HP2 tumor cells lags behind naïve KDY66 cells and FIGS.18D and 18E demonstrate that conjugated embodiments of the instantinvention immunospecifically target and kill KDY66 expressing DLL3 tumorcells but not KDY66 with DLL3 knocked down;

FIGS. 19A-19C show the ability of selected conjugated embodiments of thepresent invention to kill and/or suppress growth of exemplary lungtumorigenic cells in vivo;

FIGS. 20A-20F depict the ability of conjugated modulators of the instantinvention to substantially eradicate tumors and prevent tumor recurrencein vivo—achieving durable remissions in immunodeficient mice engraftedwith exemplary ovarian (FIG. 20A), lung (FIGS. 20B-20D) and kidneytumors (FIGS. 20E and 20F); and

FIGS. 21A-21F demonstrate that conjugated modulators of the instantinvention reduce the frequency of cancer stem cells as determined by alimiting dilution assay (LDA) for two exemplary small cell lung tumors,LU95 (FIGS. 21A-21C) and LU64 (FIGS. 21D-21F) where FIGS. 21A and 21Dshow the effect of the conjugates on tumor growth, FIGS. 21B and 21Eshow the results of the LDA and FIGS. 21C and 21F graphically presentthe reduction in cancer stem cell frequency brought about by treatmentwith the selected anti-DLL3 antibody conjugate.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

While the present invention may be embodied in many different forms,disclosed herein are specific illustrative embodiments thereof thatexemplify the principles of the invention. It should be emphasized thatthe present invention is not limited to the specific embodimentsillustrated. Moreover, any section headings used herein are fororganizational purposes only and are not to be construed as limiting thesubject matter described. Finally, for the purposes of the instantdisclosure all identifying sequence Accession numbers may be found inthe NCBI Reference Sequence (RefSeq) database and/or the NCBI GenBank®archival sequence database unless otherwise noted.

As discussed above it has surprisingly been found that DLL3 genotypicand/or phenotypic determinants are associated with various proliferativedisorders, including neoplasia exhibiting neuroendocrine features, andthat DLL3 and variants or isoforms thereof provide useful tumor markerswhich may be exploited in the treatment of related diseases. Moreover,as shown in the instant application it has unexpectedly been found thatDLL3 markers or determinants such as cell surface DLL3 protein aretherapeutically associated with cancer stem cells (also known as tumorperpetuating cells) and may be effectively exploited to eliminate orsilence the same. The ability to selectively reduce or eliminate cancerstem cells (e.g., through the use of conjugated DLL3 modulators) isparticularly surprising in that such cells are known to generally beresistant to many conventional treatments. That is, the effectiveness oftraditional, as well as more recent targeted treatment methods, is oftenlimited by the existence and/or emergence of resistant cancer stem cellsthat are capable of perpetuating tumor growth even in face of thesediverse treatment methods. Further, determinants associated with cancerstem cells often make poor therapeutic targets due to low orinconsistent expression, failure to remain associated with thetumorigenic cell or failure to present at the cell surface. In sharpcontrast to the teachings of the prior art, the instantly disclosedcompounds and methods effectively overcome this inherent resistance andto specifically eliminate, deplete, silence or promote thedifferentiation of such cancer stem cells thereby negating their abilityto sustain or re-induce the underlying tumor growth. Moreover, asexpression of DLL3 protein has largely been associated withintracellular locations such as the Golgi, it was uncertain thatphenotypic determinants could be successfully exploited as a therapeutictarget as taught herein.

Thus, it is particularly remarkable that DLL3 modulators such as thosedisclosed herein may advantageously be used in the prognosis, diagnosis,theragnosis, treatment and/or prevention of selected proliferative(e.g., neoplastic) disorders in subjects in need thereof. It will beappreciated that, while preferred embodiments of the invention will bediscussed extensively below, particularly in terms of particulardomains, regions or epitopes or in the context of cancer stem cells ortumors comprising neuroendocrine features and their interactions withthe disclosed modulators, those skilled in the art will appreciate thatthe scope of the instant invention is not limited by such exemplaryembodiments. Rather, the most expansive embodiments of the presentinvention and the appended claims are broadly and expressly directed toDLL3 modulators (including conjugated modulators) and their use in theprognosis, diagnosis, theragnosis, treatment and/or prevention of avariety of DLL3 associated or mediated disorders, including neoplasticor cell proliferative disorders, regardless of any particular mechanismof action or specifically targeted tumor, cellular or molecularcomponent.

To that end, and as demonstrated in the instant application, it hasunexpectedly been found that the disclosed DLL3 modulators caneffectively be used to target and eliminate or otherwise incapacitateproliferative or tumorigenic cells and treat DLL3 associated disorders(e.g., neoplasia). As used herein a “DLL3 associated disorder” shall beheld to mean any disorder or disease (including proliferative disorders)that is marked, diagnosed, detected or identified by a phenotypic orgenotypic aberration of DLL3 genetic components or expression (“DLL3determinant”) during the course or etiology of the disease or disorder.In this regard a DLL3 phenotypic aberration or determinant may, forexample, comprise elevated or depressed levels of DLL3 proteinexpression, abnormal DLL3 protein expression on certain definable cellpopulations or abnormal DLL3 protein expression at an inappropriatephase or stage of a cell lifecycle. Of course, it will be appreciatedthat similar expression patterns of genotypic determinants (e.g., mRNAtranscription levels) of DLL3 may also be used to classify, detect ortreat DLL3 disorders.

As used herein the term “determinant” or “DLL3 determinant” shall meanany detectable trait, property, marker or factor that is identifiablyassociated with, or specifically found in or on a particular cell, cellpopulation or tissue including those identified in or on a tissue, cellor cell population affected by a DLL3 associated disease or disorder. Inselected preferred embodiments the DLL3 modulators may associate, bindor react directly with the DLL3 determinant (e.g., cell surface DLL3protein or DLL3 mRNA) and thereby ameliorate the disorder. Moregenerally determinants may be morphological, functional or biochemicalin nature and may be genotypic or phenotypic. In other preferredembodiments the determinant is a cell surface antigen or geneticcomponent that is differentially or preferentially expressed (or is not)by specific cell types (e.g., cancer stem cells) or by cells undercertain conditions (e.g., during specific points of the cell cycle orcells in a particular niche). In still other preferred embodiments thedeterminant may comprise a gene or genetic entity that is differentlyregulated (up or down) in a specific cell or discrete cell population, agene that is differentially modified with regard to its physicalstructure and chemical composition or a protein or collection ofproteins physically associated with a gene that show differentialchemical modifications. Determinants contemplated herein arespecifically held to be positive or negative and may denote a cell, cellsubpopulation or tissue (e.g., tumors) by its presence (positive) orabsence (negative).

In a similar vein “DLL3 modulators” of the invention broadly compriseany compound that recognizes, reacts, competes, antagonizes, interacts,binds, agonizes, or associates with a DLL3 variant or isoform (orspecific domains, regions or epitopes thereof) or its genetic component.By these interactions, the DLL3 modulators may advantageously eliminate,reduce or moderate the frequency, activity, recurrence, metastasis ormobility of tumorigenic cells (e.g., tumor perpetuating cells or cancerstem cells). Exemplary modulators disclosed herein comprise nucleotides,oligonucleotides, polynucleotides, peptides or polypeptides. In certainpreferred embodiments the selected modulators will comprise antibodiesto a DLL3 protein isoform or immunoreactive fragments or derivativesthereof. Such antibodies may be antagonistic or agonistic in nature andmay optionally be conjugated or associated with a therapeutic ordiagnostic agent. Moreover, such antibodies or antibody fragments maycomprise depleting, neutralizing or internalizing antibodies. In otherembodiments, modulators within the instant invention will constitute aDLL3 construct comprising a DLL3 isoform or a reactive fragment thereof.It will be appreciated that such constructs may comprise fusion proteinsand can include reactive domains from other polypeptides such asimmunoglobulins or biological response modifiers. In still otheraspects, the DLL3 modulator will comprise a nucleic acid moiety (e.g.miRNA, siRNA, shRNA, antisense constructs, etc.) that exerts the desiredeffects at a genomic level. Still other modulators compatible with theinstant teachings will be discussed in detail below.

More generally DLL3 modulators of the present invention broadly compriseany compound that recognizes, reacts, competes, antagonizes, interacts,binds, agonizes, or associates with a DLL3 determinant (genotypic orphenotypic) including cell surface DLL3 protein. Whichever form ofmodulator is ultimately selected it will preferably be in an isolatedand purified state prior to introduction into a subject. In this regardthe term “isolated DLL3 modulator” or “isolated DLL3 antibody” shall beconstrued in a broad sense and in accordance with standardpharmaceutical practice to mean any preparation or compositioncomprising the modulator in a state substantially free of unwantedcontaminants (biological or otherwise). Moreover these preparations maybe purified and formulated as desired using various art-recognizedtechniques. Of course, it will be appreciated that such “isolated”preparations may be intentionally formulated or combined with inert oractive ingredients as desired to improve the commercial, manufacturingor therapeutic aspects of the finished product and providepharmaceutical compositions. In a broader sense the same generalconsiderations may be applied to an “isolated” DLL3 isoform or variantor an “isolated” nucleic acid encoding the same.

Further, it has surprisingly been found that modulators interacting,associating or binding to particular DLL3 domains, motifs or epitopesare especially effective in eliminating tumorigenic cells and/orsilencing or attenuating cancer stem cell influences on tumor growth orpropagation. That is, while modulators that react or associate withdomains that are proximal to the cell surface (e.g., one of the EGF-likedomains) are effective in depleting or neutralizing tumorigenic cells ithas unexpectedly been discovered that modulators associating or bindingto domains, motifs or regions that are relatively more distal to thecell surface are also effective in eliminating, neutralizing, depletingor silencing tumorigenic cells. In particular, and as shown in theappended Examples, it has been discovered that modulators that react,associate or bind to the DSL or N-terminal regions of the DLL3 proteinare surprisingly effective at eliminating or neutralizing tumorigeniccells including those exhibiting neuroendocrine features and/or cancerstem cells. This is especially true of conjugated modulators such as,for example, anti-DLL3 antibody drug conjugates comprising a cytotoxicagent. As such, it will be appreciated that certain preferredembodiments of the instant invention are directed to compounds,compositions and methods that comprise DLL3 modulators which associate,bind or react with a relatively distal portion of DLL3 including the DSLdomain and the N-terminal region.

While the present invention expressly contemplates the use of any DLL3modulator in the treatment of any DLL3 disorder, including any type ofneoplasia. In particularly preferred embodiments the disclosedmodulators may be used to prevent, treat or diagnose tumors comprisingneuroendocrine features (genotypic or phenotypic) includingneuroendocrine tumors. True or “canonical neuroendocrine tumors” (NETs)arise from the dispersed endocrine system and are typically highlyaggressive. Neuroendoerine tumors occur in the kidney, genitourinarytract (bladder, prostate, ovary, cervix, and endometrium),gastrointestinal tract (stomach, colon), thyroid (medullary thyroidcancer), and lung (small cell lung carcinoma and large cellneuroendocrine carcinoma). Moreover, the disclosed modulators mayadvantageously be used to treat, prevent or diagnose pseudoneuroendocrine tumors (pNETs) that genotypically or phenotypicallymimic, comprise, resemble or exhibit common traits with canonicalneuroendocrine tumors. “Pseudo neuroendocrine tumors” are tumors thatarise from cells of the diffuse neuroendocrine system or from cells inwhich a neuroendocrine differentiation cascade has been aberrantlyreactivated during the oncogenic process. Such pNETs commonly sharecertain genotypic, phenotypic or biochemical characteristics withtraditionally defined neuroendocrine tumors, including the ability toproduce subsets of biologically active amines, neurotransmitters, andpeptide hormones. Accordingly, for the purposes of the instant inventionthe phrases “tumors comprising neuroendocrine features” or “tumorsexhibiting neuroendocrine features” shall be held to comprise bothneuroendocrine tumors and pseudo neuroendocrine tumors unless otherwisedictated by context.

Besides the association with tumors generally discussed above, there arealso indications of phenotypic or genotypic association between selectedtumor initiating cells (TIC) and DLL3 determinants. In this regardselected TICs (e.g., cancer stem cells) may express elevated levels ofDLL3 protein when compared to normal tissue and non-tumorigenic cells(NTG), which together typically comprise much of a solid tumor. Thus,DLL3 determinants may comprise a tumor associated marker (or antigen orimmunogen) and the disclosed modulators may provide effective agents forthe detection and suppression of TIC and associated neoplasia due toaltered levels of the proteins on cell surfaces or in the tumormicroenvironment. Accordingly, DLL3 modulators, including immunoreactiveantagonists and antibodies that associate, bind or react with theproteins, may effectively reduce the frequency of tumor initiating cellsand could be useful in eliminating, depleting, incapacitating, reducing,promoting the differentiation of, or otherwise precluding or limitingthe ability of these tumor-initiating cells to lie dormant and/orcontinue to fuel tumor growth, metastasis or recurrence in a patient. Inthis regard those skilled in the art will appreciate that the presentinvention further provides DLL3 modulators and their use in reducing thefrequency of tumor initiating cells.

II. DLL3 Physiology

The Notch signaling pathway, first identified in C. elegans andDrosophila and subsequently shown to be evolutionarily conserved frominvertebrates to vertebrates, participates in a series of fundamentalbiological processes including normal embryonic development, adulttissue homeostasis, and stem cell maintenance (D'Souza et al., 2010; Liuet al., 2010). Notch signaling is critical for a variety of cell typesduring specification, patterning and morphogenesis. Frequently, thisoccurs through the mechanism of lateral inhibition, in which cellsexpressing Notch ligand(s) adopt a default cell fate, yet suppress thisfate in adjacent cells via stimulation of Notch signaling (Sternberg,1988, Cabrera 1990). This binary cell fate choice mediated by Notchsignaling is found to play a role in numerous tissues, including thedeveloping nervous system (de la Pompa et al., 1997), the hematopoieticand immune systems (Bigas and Espinosoa, 2012; Hoyne et al, 2011; Nagaseet al., 2011), the gut (Fre et al., 2005; Fre et al., 2009), theendocrine pancreas (Apelqvist et al., 1999; Jensen et al., 2000), thepituitary (Raetzman et al., 2004), and the diffuse neuroendocrine system(Ito et al., 2000; Schonhoff et al, 2004). A generalized mechanism forimplementing this binary switch appears conserved despite the wide rangeof developmental systems in which Notch plays a role—in cells where thedefault cell fate choice is determined by transcriptional regulatorsknown as basic helix-loop-helix (bHLH) proteins, Notch signaling leadsto activation of a class of Notch responsive genes, which in turnsuppress the activity of the bHLH proteins (Ball, 2004). These binarydecisions take place in the wider context of developmental and signalingcues that permit Notch signaling to effect proliferation or inhibit it,and to trigger self-renewal or inhibit it.

In Drosophila, Notch signaling is mediated primarily by one Notchreceptor gene and two ligand genes, known as Serrate and Delta (Whartonet al, 1985; Rebay et al., 1991). In humans, there are four known Notchreceptors and five DSL (Delta-Serrate LAG2) ligands—two homologs ofSerrate, known as Jagged1 and Jagged 2, and three homologs of Delta,termed delta-like ligands or DLL1, DLL3 and DLL4. In general, Notchreceptors on the surface of the signal-receiving cell are activated byinteractions with ligands expressed on the surface of an opposing,signal-sending cell (termed a trans-interaction). Thesetrans-interactions lead to a sequence of protease mediated cleavages ofthe Notch receptor. In consequence, the Notch receptor intracellulardomain is free to translocate from the membrane to the nucleus, where itpartners with the CSL family of transcription factors (RBPJ in humans)and converts them from transcriptional repressors into activators ofNotch responsive genes.

Of the human Notch ligands, DLL3 is different in that it seems incapableof activating the Notch receptor via trans-interactions (Ladi et al.,2005). Notch ligands may also interact with Notch receptors in cis (onthe same cell) leading to inhibition of the Notch signal, although theexact mechanisms of cis-inhibition remain unclear and may vary dependingupon the ligand (for instance, see Klein et al., 1997; Ladi et al.,2005; Glittenberg et al., 2006). Two hypothesized modes of inhibitioninclude modulating Notch signaling at the cell surface by preventingtrans-interactions, or by reducing the amount of Notch receptor on thesurface of the cell by perturbing the processing of the receptor or byphysically causing retention of the receptor in the endoplasmicreticulum or Golgi (Sakamoto et al., 2002; Dunwoodle, 2009). It isclear, however, that stochastic differences in expression of Notchreceptors and ligands on neighboring cells can be amplified through bothtranscriptional and non-transcriptional processes, and subtle balancesof cis- and trans-interactions can result in a fine tuning of the Notchmediated delineation of divergent cell fates in neighboring tissues(Sprinzak et al., 2010).

DLL3 (also known as Delta-like 3 or SCDO1) is a member of the Delta-likefamily of Notch DSL ligands. Representative DLL3 protein orthologsinclude, but are not limited to, human (Accession Nos. NP_058637 andNP_982353), chimpanzee (Accession No. XP_00331639), mouse (Accession No.NP_031892), and rat (Accession No. NP_446118). In humans, the DLL3 geneconsists of 8 exons spanning 9.5 kBp located on chromosome 19q13.Alternate splicing within the last exon gives rise to two processedtranscripts, one of 2389 bases (Accession No. NM_016941; FIG. 1A, SEQ IDNO: 1) and one of 2052 bases (Accession No. NM_203486; FIG. 1B, SEQ IDNO: 2). The former transcript encodes a 618 amino acid protein(Accession No. NP_058637; FIG. 1C, SEQ ID NO: 3), whereas the latterencodes a 587 amino acid protein (Accession No. NP_982353; FIG. 1D, SEQID NO: 4). These two protein isoforms of DLL3 share overall 100%identity across their extracellular domains and their transmembranedomains, differing only in that the longer isoform contains an extendedcytoplasmic tail containing 32 additional residues at the carboxyterminus of the protein (FIG. 1E). The biological relevance of theisoforms is unclear, although both isoforms can be detected in tumorcells (FIG. 5). Percent identities for each of the members of thedelta-like family of proteins in humans are shown in FIG. 2A, as well ascross species identities in FIG. 2B.

In general, DSL ligands are composed of a series of structural domains:a unique N-terminal domain, followed by a conserved DSL domain, multipletandem epidermal growth factor (EGF)-like repeats, a transmembranedomain, and a cytoplasmic domain not highly conserved across ligands butone which contains multiple lysine residues that are potential sites forubiquitination by unique E3 ubiquitin ligases. The DSL domain is adegenerate EGF-domain that is necessary but not sufficient forinteractions with Notch receptors (Shimizu et al., 1999). Additionally,the first two EGF-like repeats of most DSL ligands contain a smallerprotein sequence motif known as a DOS domain that co-operativelyinteracts with the DSL domain when activating Notch signaling.

FIG. 1F provides a schematic diagram of the extracellular region of theDLL3 protein, illustrating the general juxtaposition of the six EGF-likedomains, the single DSL domain and the N-terminal domain. Generally, theEGF domains are recognized as occurring at about amino acid residues216-249 (domain 1), 274-310 (domain 2), 312-351 (domain 3), 353-389(domain 4), 391-427 (domain 5) and 429-465 (domain 6), with the DSLdomain at about amino acid residues 176-215 and the N-terminal domain atabout amino acid residues 27-175 of hDLL3 (SEQ ID NOS: 3 and 4). Asdiscussed in more detail herein and shown in Example 10 below, each ofthe EGF-like domains, the DSL domain and the N-terminal domain comprisepart of the DLL3 protein as defined by a distinct amino acid sequence.Note that, for the purposes of the instant disclosure the respectiveEGF-like domains may be termed EGF1. to EGF6 with EGF1 being closest tothe N-terminal portion of the protein. In regard to the structuralcomposition of the protein one significant aspect of the instantinvention is that the disclosed DLL3 modulators may be generated,fabricated, engineered or selected so as to react with a selecteddomain, motif or epitope. In certain cases such site specific modulatorsmay provide enhanced reactivity and/or efficacy depending on theirprimary mode of action.

Note that, as used herein the terms “mature protein” or “maturepolypeptide” as used herein refers to the form(s) of the proteinproduced by expression in a mammalian cell. It is generally hypothesizedthat once export of a growing protein chain across the rough endoplasmicreticulum has been initiated, proteins secreted by mammalian cells havea signal peptide (SP) sequence which is cleaved from the completepolypeptide to produce a “mature” form of the protein. In both isoformsof DLL3 the mature protein comprises a signal peptide of 26 amino acidsthat may be clipped prior to cell surface expression. Thus, in matureproteins the N-terminal domain will extend from position 27 in theprotein until the beginning of the DSL domain. Of course, if the proteinis not processed in this manner the N-terminal domain would be held toextend to position one of SEQ ID NOS: 3 & 4.

Of the various Delta-like ligands, DLL3 is the most divergent from theothers in the family, since it contains a degenerate DSL domain, no DOSmotifs, and an intracellular domain which lacks lysine residues. Thedegenerate DSL and lack of DOS motifs are consistent with the inabilityof DLL3 to trigger Notch signaling in trans (between cells), suggestingthat DLL3, unlike DLL1 or DLL4, acts only as an inhibitor of Notchsignaling (Ladi et al., 2005). Studies have shown that DLL3 may beresident primarily in the cis-Golgi (Geffers et al., 2007), which wouldbe consistent with a hypothesized ability to retain Notch receptorintracellularly, or to interfere with processing of Notch receptors,preventing export to the cell surface and instead retargeting it to thelysosome (Chapman et al., 2011). Some DLL3 protein may appear at thecell surface, however, when the protein is artificially overexpressed inmodel systems (Ladi et al., 2005), but it is not obvious that this wouldbe the case in normal biological contexts nor in tumors in which theDLL3 mRNA transcript is elevated; somewhat surprisingly, the proteinlevels detected in tumor types disclosed herein indicate significantDLL3 protein is escaping to the cell surface of various tumors.

Defects in the DLL3 gene have been linked to spondylocostal dysostosisin humans, a severe congenital birth defect resulting in abnormalvertebrae formation and rib abnormalities (Dunwoodie, 2009). This islinked to alterations in Notch signaling, known to play a crucial rolein determining the polarity and patterning of somites, the embryonicprecursors to the vertebrae that require a finely regulated oscillatinginterplay between Notch, Wnt, and FGF signaling pathways for properdevelopment (Kageyama et al., 2007; Goldbeter and Pourquie, 2008).Although DLL1 and DLL3 are typically expressed in similar locationswithin the developing mouse embryo, experiments with transgenic micehave demonstrated that DLL3 does not compensate for DLL1 (Geffers etal., 2007). DLL1 knock-out mice are embryonic lethal, but DLL3 mutantmice do survive yet show a phenotype similar to that found in humanswith spondylocostal dysostosis (Kusumi et al., 1998; Shinkai et al.,2004). These results data are consistent with a subtle interplay ofNotch trans- and cis-interactions crucial for normal development.

Further, as discussed above Notch signaling plays a role in thedevelopment and maintenance of neuroendocrine cells and tumorsexhibiting neuroendocrine features. In this regard Notch signaling isinvolved in a wide range of cell fate decisions in normal endocrineorgans and in the diffuse neuroendocrine system. For instance, in thepancreas, Notch signaling is required to suppress the development of adefault endocrine phenotype mediated by the bHLH transcription factorNGN3 (Habener et al, 2005). Similar Notch mediated suppression ofendocrine cell fates occurs in enteroendocrine cells (Schonhoff et al.,2004), thyroid parafollicular cells (Cook et al., 2010), in specifyingthe relative ratios of neuroendocrine cell types in the pituitary (Duttaet al., 2011), and is likely involved in decisions of cells within thelungs to adopt a neuroendocrine or non-neuroendocrine pheneotype (Chenet at., 1997; Ito et al., 2000; Sriuranpong et al., 2002). Hence it isclear that in many tissues, suppression of Notch signaling is linked toneuroendocrine phenotypes.

Inappropriate reactivation of developmental signaling pathways ordisregulation of normal signaling pathways are commonly observed intumors, and in the case of Notch signaling, have been associated withnumerous tumor types (Koch and Radtke, 2010; Harris et al., 2012). TheNotch pathway has been studied as an oncogene in lymphomas, colorectal,pancreatic, and some types of non-small cell lung cancer (see Zarenczanand Chen, 2010 and references therein). In contrast, Notch is reportedto act as a tumor suppressor in tumors with neuroendocrine features (seeZarenczan and Chen, 2010 supra). Tumors with neuroendocrine featuresarise infrequently in a wide range of primary sites, and while theirexhaustive classification remains problematic (Yao et al., 2008;Klimstra et al., 2010; Klöppel, 2011), they may be classified into fourmajor types: low grade benign carcinoids, low-grade well-differentiatedneuroendocrine tumors with malignant behavior, tumors with mixedneuroendocrine and epithelial features, and high-grade poorlydifferentiated neuroendocrine carcinomas. Of these classifications, thepoorly differentiated neuroendocrine carcinomas, which include smallcell lung cancer (SCLC) and subsets of non-small cell lung cancer(NSCLC), are cancer types with dismal prognoses. It has been postulatedthat SCLC is bronchogenic in origin, arising in part from pulmonaryneuroendocrine cells (Galluzzo and Bocchetta, 2011). Whatever thespecific cellular source of origin for each of these tumors possessing aneuroendocrine phenotype, it may be expected that suppression of Notchsignaling, either by direct lesions in the Notch pathway genesthemselves, or by activation of other genes that suppress Notchsignaling, may lead to the acquisition of the neuroendocrine phenotypeof these tumors. By extension, the genes that lead to the perturbationof the Notch pathway may afford therapeutic targets for the treatment oftumors with neuroendocrine phenotypes, particularly for indications thatcurrently have poor clinical outcomes.

ASCL1 is one such gene that appears to interact with Notch signalingpathway via DLL3. It is clear that many neuroendocrine tumors show apoorly differentiated (i.e. partially complete) endocrine phenotype; forinstance, marked elevation or expression of various endocrine proteinsand polypeptides (e.g. chromogranin A, CHGA; calcitonin, CALCA;propiomelanocorin, POMC; somatostatin, SST), proteins associated withsecretory vesicles (e.g., synaptophysin, SYP), and genes involved in thebiochemical pathways responsible for the synthesis of bioactive amines(e.g., dopa decarboxylase, DDC). Perhaps not surprisingly, these tumorsfrequently over-express ASCL1 (also known as mASH1 in mice, or hASH1 inhumans), a transcription factor known to play a role in orchestratinggene cascades leading to neural and neuroendocrine phenotypes. Althoughthe specific molecular details of the cascade remain ill-defined, it isincreasingly clear that for certain cell types, particularly thyroidparafollicular cells (Kameda et al., 2007), chromaffin cells of theadrenal medulla (Huber et al., 2002) and cells found in the diffuseneuroendocrine system of the lung (Chen et al., 1997; Ito et al., 2000;Sriuranpong et al., 2002), ASCL1 is part of a finely tuned developmentalregulatory loop in which cell fate choices are mediated by the balanceof ASCL1-mediated and Notch-mediated gene expression cascades (FIG. 3).For instance, ASCL1 was found in to be expressed in normal mousepulmonary neuroendocrine cells, while the Notch signaling effector HES1,was expressed in pulmonary non-neuroendocrine cells (Ito et al., 2000).That these two cascades are in a fine balance with potentialcross-regulation is increasingly appreciated. The Notch effector HES1has been shown to downregulate ASCL1 expression (Chen et al., 1997;Sriuranpong et al., 2002). These results clearly demonstrate that Notchsignaling can suppress neuroendocrine differentiation. However,demonstration that ASCL1 binding to the DLL3 promoter activates DLL3expression (Henke et al., 2009) and the observation that DLL3 attenuatesNotch signaling (Ladi et al., 2005) closes the genetic circuit for cellfate choices between neuroendocrine and non-neuroendocrine phenotypes.

Given that Notch signaling appears to have evolved to amplify subtledifferences between neighboring cells to permit sharply bounded tissuedomains with divergent differentiation paths (e.g., “lateralinhibition,” as described above), these data together suggest that afinely tuned developmental regulatory loop (FIG. 3) has becomereactivated and disregulated in cancers with neuroendocrine phenotypes.While it is not obvious that DLL3 would provide a suitable cell surfacetarget for the development of antibody therapeutics given its normalresidence within interior membranous compartments of the cell (Gefferset al., 2007) and its presumed interactions with Notch therein, it ispossible that the resultant elevation of DLL3 expression inneuroendocrine tumors may offer a unique therapeutic target for tumorswith the neuroendocrine phenotype (e.g., NETs and pNETs). It is commonlyobserved that vast overexpression of proteins in laboratory systems maycause mislocalization of the overexpressed protein within the cell.Therefore it is a reasonable hypothesis, yet not obvious withoutexperimental verification, that overexpression of DLL3 in tumors maylead to some cell surface expression of the protein, and thereby presenta target for the development of antibody therapeutics.

III. Cancer Stem Cells

As alluded to above it has surprisingly been discovered that aberrantDLL3 expression (genotypic and/or phenotypic) is associated with varioustumorigenic cell subpopulations. In this respect the present inventionprovides DLL3 modulators that may be particularly useful for targetingsuch cells, and especially tumor perpetuating cells, therebyfacilitating the treatment, management or prevention of neoplasticdisorders. Thus, in preferred embodiments modulators of DLL3determinants (phenotypic or genotypic) may be advantageously be used toreduce tumor initiating cell frequency in accordance with the presentteachings and thereby facilitate the treatment or management ofproliferative disorders.

For the purposes of the instant application the term “tumor initiatingcell” (TIC) encompasses both “tumor perpetuating cells” (TPC; i.e.,cancer stem cells or CSC) and highly proliferative “tumor progenitorcells” (termed TProg), which together generally comprise a uniquesubpopulation (i.e. 0.1-40%) of a bulk tumor or mass. For the purposesof the instant disclosure the terms “tumor perpetuating cells” and“cancer stem cells” or “neoplastic stem cells” are equivalent and may beused interchangeably herein. TPC differ from TProg in that TPC cancompletely recapitulate the composition of tumor cells existing within atumor and have unlimited self-renewal capacity as demonstrated by serialtransplantation (two or more passages through mice) of low numbers ofisolated cells, whereas TProg will not display unlimited self-renewalcapacity.

Those skilled in the art will appreciate that fluorescence-activatedcell sorting (FACS) using appropriate cell surface markers is a reliablemethod to isolate highly enriched cancer stem cell subpopulations(e.g., >99.5% purity) due, at least in part, to its ability todiscriminate between single cells and clumps of cells (i.e. doublets,etc.). Using such techniques it has been shown that when low cellnumbers of highly purified TProg cells are transplanted intoimmunocompromised mice they can fuel tumor growth in a primarytransplant. However, unlike purified TPC subpopulations the TProggenerated tumors do not completely reflect the parental tumor inphenotypic cell heterogeneity and are demonstrably inefficient atreinitiating serial tumorigenesis in subsequent transplants. Incontrast, TPC subpopulations completely reconstitute the cellularheterogeneity of parental tumors and can efficiently initiate tumorswhen serially isolated and transplanted. Thus, those skilled in the artwill recognize that a definitive difference between TPC and TProg,though both may be tumor generating in primary transplants, is theunique ability of TPC to perpetually fuel heterogeneous tumor growthupon serial transplantation at low cell numbers. Other common approachesto characterize TPC involve morphology and examination of cell surfacemarkers, transcriptional profile, and drug response although markerexpression may change with culture conditions and with cell line passagein vitro.

Accordingly, for the purposes of the instant invention tumorperpetuating cells, like normal stem cells that support cellularhierarchies in normal tissue, are preferably defined by their ability toself-renew indefinitely while maintaining the capacity for multilineagedifferentiation. Tumor perpetuating cells are thus capable of generatingboth tumorigenic progeny (i.e., tumor initiating cells: TPC and TProg)and non-tumorigenic (NTG) progeny. As used herein a “non-tumorigeniccell” (NTG) refers to a tumor cell that arises from tumor initiatingcells, but does not itself have the capacity to self-renew or generatethe heterogeneous lineages of tumor cells that comprise a tumor.Experimentally, NTG cells are incapable of reproducibly forming tumorsin mice, even when transplanted in excess cell numbers.

As indicated, TProg are also categorized as tumor initiating cells (orTIC) due to their limited ability to generate tumors in mice. TProg areprogeny of TPC and are typically capable of a finite number ofnon-self-renewing cell divisions. Moreover, TProg cells may further bedivided into early tumor progenitor cells (ETP) and late tumorprogenitor cells (LTP), each of which may be distinguished by phenotype(e.g., cell surface markers) and different capacities to recapitulatetumor cell architecture. In spite of such technical differences, bothETP and LTP differ functionally from TPC in that they are generally lesscapable of serially reconstituting tumors when transplanted at low cellnumbers and typically do not reflect the heterogeneity of the parentaltumor. Notwithstanding the foregoing distinctions, it has also beenshown that various TProg populations can, on rare occasion, gainself-renewal capabilities normally attributed to stem cells andthemselves become TPC (or CSC). In any event both types oftumor-initiating cells are likely represented in the typical tumor massof a single patient and are subject to treatment with the modulators asdisclosed herein. That is, the disclosed compositions are generallyeffective in reducing the frequency or altering the chemosensitivity ofsuch DLL3 positive tumor initiating cells regardless of the particularembodiment or mix represented in a tumor.

In the context of the instant invention, TPC are more tumorigenic,relatively more quiescent and often more chemoresistant than the TProg(both ETP and LTP), NTG cells and the tumor-infiltrating non-TPC derivedcells (e.g., fibroblasts/stroma, endothelial & hematopoietic cells) thatcomprise the bulk of a tumor. Given that conventional therapies andregimens have, in large part, been designed to both debulk tumors andattack rapidly proliferating cells, TPC are likely to be more resistantto conventional therapies and regimens than the faster proliferatingTProg and other bulk tumor cell populations. Further, TPC often expressother characteristics that make them relatively chemoresistant toconventional therapies, such as increased expression of multi-drugresistance transporters, enhanced DNA repair mechanisms andanti-apoptotic proteins. These properties, each of which contribute todrug tolerance by TPC, constitute a key reason for the failure ofstandard oncology treatment regimens to ensure long-term benefit formost patients with advanced stage neoplasia; i.e. the failure toadequately target and eradicate those cells that fuel continued tumorgrowth and recurrence (i.e. TPC or CSC).

Unlike many prior art treatments, the novel compositions of the presentinvention preferably reduce the frequency of tumor initiating cells uponadministration to a subject regardless of the form or specific target(e.g., genetic material, DLL3 antibody or ligand fusion construct) ofthe selected modulator. As noted above, the reduction in tumorinitiating cell frequency may occur as a result of a) elimination,depletion, sensitization, silencing or inhibition of tumor initiatingcells; b) controlling the growth, expansion or recurrence of tumorinitiating cells; c) interrupting the initiation, propagation,maintenance, or proliferation of tumor initiating cells; or d) byotherwise hindering the survival, regeneration and/or metastasis of thetumorigenic cells. In some embodiments, the reduction in the frequencyof tumor initiating cells occurs as a result of a change in one or morephysiological pathways. The change in the pathway, whether by reductionor elimination of the tumor initiating cells or by modifying theirpotential (e.g., induced differentiation, niche disruption) or otherwiseinterfering with their ability to influence the tumor environment orother cells, in turn allows for the more effective treatment of DLL3associated disorders by inhibiting tumorigenesis, tumor maintenanceand/or metastasis and recurrence.

Among art-recognized methods that can be used to assess such a reductionin the frequency of tumor initiating cells is limiting dilution analysiseither in vitro or in vivo, preferably followed by enumeration usingPoisson distribution statistics or assessing the frequency of predefineddefinitive events such as the ability to generate tumors in vivo or not.While such limiting dilution analysis comprise preferred methods ofcalculating reduction of tumor initiating cell frequency other, lessdemanding methods, may also be used to effectively determine the desiredvalues, albeit slightly less accurately, and are entirely compatiblewith the teachings herein. Thus, as will be appreciated by those skilledin the art, it is also possible to determine reduction of frequencyvalues through well-known flow cytometric or immunohistochemical means.As to all the aforementioned methods see, for example, Dylla et al.2008, PMID: 18560594 & Hoey et al. 2009, PMID: 19664991; each of whichis incorporated herein by reference in its entirety and, in particular,for the disclosed methods.

With respect to limiting dilution analysis, in vitro enumeration oftumor initiating cell frequency may be accomplished by depositing eitherfractionated or unfractionated human tumor cells (e.g. from treated anduntreated tumors, respectively) into in vitro growth conditions thatfoster colony formation. In this manner, colony forming cells might beenumerated by simple counting and characterization of colonies, or byanalysis consisting of, for example, the deposition of human tumor cellsinto plates in serial dilutions and scoring each well as either positiveor negative for colony formation at least 10 days after plating. In vivolimiting dilution experiments or analyses, which are generally moreaccurate in their ability to determine tumor initiating cell frequencyencompass the transplantation of human tumor cells, from eitheruntreated control or treated populations, for example, intoimmunocompromised mice in serial dilutions and subsequently scoring eachmouse as either positive or negative for tumor formation at least 60days after transplant. The derivation of cell frequency values bylimiting dilution analysis in vitro or in vivo is preferably done byapplying Poisson distribution statistics to the known frequency ofpositive and negative events, thereby providing a frequency for eventsfulfilling the definition of a positive event; in this case, colony ortumor formation, respectively.

As to other methods compatible with the instant invention that may beused to calculate tumor initiating cell frequency, the most commoncomprise quantifiable flow cytometric techniques and immunohistochemicalstaining procedures. Though not as precise as the limiting dilutionanalysis techniques described immediately above, these procedures aremuch less labor intensive and provide reasonable values in a relativelyshort time frame. Thus, it will be appreciated that a skilled artisanmay use flow cytometric cell surface marker profile determinationemploying one or more antibodies or reagents that bind art-recognizedcell surface proteins known to enrich for tumor initiating cells (e.g.,potentially compatible markers as are set forth in PCT application2012/031280 which is incorporated herein in its entirety) and therebymeasure TIC levels from various samples. In still another compatiblemethod one skilled in the art might enumerate TIC frequency in situ(e.g., in a tissue section) by immunohistochemistry using one or moreantibodies or reagents that are able to bind cell surface proteinsthought to demarcate these cells.

Those skilled in the art will recognize that numerous markers (or theirabsence) have been associated with various populations of cancer stemcells and used to isolate or characterize tumor cell subpopulations. Inthis respect exemplary cancer stem cell markers comprise OCT4, Nanog,STAT3, EPCAM, CD24, CD34, NB84, TrkA, GD2, CD133, CD20, CD56, CD29,B7H3, CD46, transferrin receptor, JAM3, carboxypeptidase M, ADAM9,oncostatin M, Lgr5, Lgr6, CD324, CD325, nestin, Sox1, Bmi-1, eed,easyh1, easyh2, mf2, yy1, smarcA3, smarckA5, smarcD3, smarcE1, mllt3,FZD1, FZD2, FZD3, FZD4, FZD6, FZD7, FZD8, FZD9, FZD10, WNT2, WNT2B,WNT3, WNT5A, WNT10B, WNT16, AXIN1, BCL9, MYC, (TCF4) SLC7A8, IL1RAP,TEM8, TMPRSS4, MUC16, GPRC5B, SLC6A14, SLC4A11, PPAP2C, CAV1, CAV2,PTPN3, EPHA1, EPHA2, SLC1A1, CX3CL1, ADORA2A, MPZL1, FLJ10052, C4.4A,EDG3, RARRES1, TMEPALI, PTS, CEACAM6, NID2, STEAP, ABCA3, CRIM1, IL1R1,OPN3, DAF, MUC1, MCP, CPD, NMA, ADAM9, GJA1, SLC19A2, ABCA1, PCDH7,ADCY9, SLC39A1, NPC1, ENPP1, N33, GPNMB, LY6E, CELSR1, LRP3, C20orf52,TMEPA1, FLVCR, PCDHA10, GPR54, TGFBR3, SEMA4B, PCDHB2, ABCG2, CD166,AFP, BMP-4, β-catenin, CD2, CD3, CD9, CD14, CD31, CD38, CD44, CD45,CD74, CD90, CXCR4, decorin, EGFR, CD105, CD64, CD16, CD16a, CD16b, GLI1,GL12, CD49b, and CD49f. See, for example, Schulenburg et al., 2010,PMID: 20185329, U.S. Pat. No. 7,632,678 and U.S.P.Ns. 2007/0292414,2008/0175870, 2010/0275280, 2010/0162416 and 2011/0020221 each of whichis incorporated herein by reference. It will further be appreciated thateach of the aforementioned markers may also be used as a secondarytarget antigen in the context of the bispecific or multispecificantibodies of the instant invention.

Similarly, non-limiting examples of cell surface phenotypes associatedwith cancer stem cells of certain tumor types include CD44^(hi)CD24^(low), ALDH⁺, CD133⁺, CD123⁺, CD34⁺CD38⁻, CD44⁺CD24⁻,CD46^(hi)CD324⁺CD66c⁻, CD133⁺CD34⁺CD10⁻CD1⁻, CD138⁻CD34⁻CD19⁺,CD133⁺RC2⁺, CD44⁺α₂ β₁ ^(hi)CD133⁺, CD44⁺CD24⁺ESA⁺, CD271⁺, ABCB5⁺ aswell as other cancer stem cell surface phenotypes that are known in theart. See, for example, Schulenburg et al., 2010, supra, Visvader et al.,2008, PMID: 18784658 and U.S.P.N. 2008/0138313, each of which isincorporated herein in its entirety by reference. Those skilled in theart will appreciate that marker phenotypes such as those exemplifiedimmediately above may be used in conjunction with standard flowcytometric analysis and cell sorting techniques to characterize,isolate, purify or enrich TIC and/or TPC cells or cell populations forfurther analysis. Of interest with regard to the instant invention CD46,CD324 and, optionally, CD66c are either highly or heterogeneouslyexpressed on the surface of many human colorectal (“CR”), breast (“BR”),non-small cell lung (NSCLC), small cell lung (SCLC), pancreatic (“PA”),melanoma (“Mel”), ovarian (“OV”), and head and neck cancer (“HN”) tumorcells, regardless of whether the tumor specimens being analyzed wereprimary patient tumor specimens or patient-derived NTX tumors.

Using any of the above-referenced methods and selected markers as knownin the art (and shown in Example 17 below) it is then possible toquantify the reduction in frequency of TIC (or the TPC therein) providedby the disclosed DLL3 modulators (including those conjugated tocytotoxic agents) in accordance with the teachings herein. In someinstances, the compounds of the instant invention may reduce thefrequency of TIC or TPC (by a variety of mechanisms noted above,including elimination, induced differentiation, niche disruption,silencing, etc.) by 10%, 15%, 20%, 25%, 30% or even by 35%. In otherembodiments, the reduction in frequency of TIC or TPC may be on theorder of 40%, 45%, 50%, 55%, 60% or 65%. In certain embodiments, thedisclosed compounds my reduce the frequency of TIC or TPC by 70%, 75%,80%, 85%, 90% or even 95%. Of course it will be appreciated that anyreduction of the frequency of the TIC or TPC likely results in acorresponding reduction in the tumorigenicity, persistence, recurrenceand aggressiveness of the neoplasia.

IV. DLL3 Modulators

In any event, the present invention is directed to the use of DLL3modulators, including DLL3 antagonists, for the diagnosis, theragnosis,treatment and/or prophylaxis of various disorders including any one of anumber of DLL3 associated malignancies. The disclosed modulators may beused alone or in conjunction with a wide variety of anti-cancercompounds such as chemotherapeutic or immunotherapeutic agents (e.g.,therapeutic antibodies) or biological response modifiers. In otherselected embodiments, two or more discrete DLL3 modulators may be usedin combination to provide enhanced anti-neoplastic effects or may beused to fabricate multispecific constructs.

In certain embodiments, the DLL3 modulators of the present inventionwill comprise nucleotides, oligonucleotides, polynucleotides, peptidesor polypeptides. More particularly, exemplary modulators of theinvention may comprise antibodies and antigen-binding fragments orderivatives thereof, proteins, peptides, glycoproteins, glycopeptides,glycolipids, polysaccharides, oligosaccharides, nucleic acids, antisenseconstructs, siRNA, miRNA, bioorganic molecules, peptidomimetics,pharmacological agents and their metabolites, transcriptional andtranslation control sequences, and the like. In certain embodiments themodulators will comprise soluble DLL3 (sDLL3) or a form, variant,derivative or fragment thereof including, for example, DLL3 fusionconstructs (e.g., DLL3-Fc, DLL3-targeting moiety, etc.) orDLL3-conjugates (e.g., DLL3-PEG, DLL3-cytotoxic agent, DLL3-brm, etc.).In other preferred embodiments the DLL3 modulators comprise antibodiesor immunoreactive fragments or derivatives thereof. In particularlypreferred embodiments the modulators of the instant invention willcomprise neutralizing, depleting or internalizing antibodies orderivatives or fragments thereof. Moreover, as with the aforementionedfusion constructs, such antibody modulators may be conjugated, linked orotherwise associated with selected cytotoxic agents, polymers,biological response modifiers (BRMs) or the like to provide directedimmunotherapies with various (and optionally multiple) mechanisms ofaction. As alluded to above such antibodies may be pan-DLL antibodiesand associate with two or more DLL family members or, in thealternative, comprise antigen binding molecules that selectively reactwith one or both isoforms of DLL3. In yet other preferred embodimentsthe modulators may operate on the genetic level and may comprisecompounds as antisense constructs, siRNA, miRNA and the like thatinteract or associate with the genotypic component of a DLL3determinant.

It will further be appreciated that the disclosed DLL3 modulators maydeplete, silence, neutralize, eliminate or inhibit growth, propagationor survival of tumor cells, including TPC, and/or associated neoplasiathrough a variety of mechanisms, including agonizing or antagonizingselected pathways or eliminating specific cells depending, for example,on the form of DLL3 modulator, any associated payload or dosing andmethod of delivery. Thus, while preferred embodiments disclosed hereinare directed to the depletion, inhibition or silencing of specific tumorcell subpopulations such as tumor perpetuating cells or to modulatorsthat interact with a specific epitope or domain, it must be emphasizedthat such embodiments are merely illustrative and not limiting in anysense. Rather, as set forth in the appended claims, the presentinvention is broadly directed to DLL3 modulators and their use in thetreatment, management or prophylaxis of various DLL3 associateddisorders irrespective of any particular mechanism, binding region ortarget tumor cell population.

Regardless of the form of the modulator selected it will be appreciatedthat the chosen compound may be antagonistic in nature. As used hereinan “antagonist” refers to a molecule capable of neutralizing, blocking,inhibiting, abrogating, reducing or interfering with the activities of aparticular or specified target (e.g., DLL3), including the binding ofreceptors to ligands or the interactions of enzymes with substrates. Inthis respect it will be appreciated that DLL3 antagonists of the instantinvention may comprise any ligand, polypeptide, peptide, fusion protein,antibody or immunologically active fragment or derivative thereof thatrecognizes, reacts, binds, combines, competes, associates or otherwiseinteracts with the DLL3 protein or fragment thereof and eliminates,silences, reduces, inhibits, hinders, restrains or controls the growthof tumor initiating cells or other neoplastic cells including bulk tumoror NTG cells. Compatible antagonists may further include small moleculeinhibitors, aptamers, antisense constructs, siRNA, miRNA and the like,receptor or ligand molecules and derivatives thereof which recognize orassociate with a DLL3 genotypic or phenotypic determinant therebyaltering expression patterns or sequestering its binding or interactionwith a substrate, receptor or ligand.

As used herein and applied to two or more molecules or compounds, theterms “recognizes” or “associates” shall be held to mean the reaction,binding, specific binding, combination, interaction, connection,linkage, uniting, coalescence, merger or joining, covalently ornon-covalently, of the molecules whereby one molecule exerts an effecton the other molecule.

Moreover, as demonstrated in the examples herein (e.g., see FIG. 2B),some modulators of human DLL3 may, in certain cases, cross-react withDLL3 from a species other than human (e.g., murine). In other casesexemplary modulators may be specific for one or more isoforms of humanDLL3 and will not exhibit cross-reactivity with DLL3 orthologs fromother species. Of course, in conjunction with the teachings herein suchembodiments may comprise pan-DLL antibodies that associate with two ormore DLL family members from a single species or antibodies thatexclusively associate with DLL3.

In any event, and as will be discussed in more detail below, thoseskilled in the art will appreciate that the disclosed modulators may beused in a conjugated or unconjugated form. That is, the modulator may beassociated with or conjugated to (e.g. covalently or non-covalently)pharmaceutically active compounds, biological response modifiers,anti-cancer agents, cytotoxic or cytostatic agents, diagnostic moietiesor biocompatible modifiers. In this respect it will be understood thatsuch conjugates may comprise peptides, polypeptides, proteins, fusionproteins, nucleic acid molecules, small molecules, mimetic agents,synthetic drugs, inorganic molecules, organic molecules andradioisotopes. Moreover, as indicated herein the selected conjugate maybe covalently or non-covalently linked to the DLL3 modulator in variousmolar ratios depending, at least in part, on the method used to effectthe conjugation.

V. Modulator Fabrication and Supply

A. Antibody Modulators

1. Overview

As previously alluded to particularly preferred embodiments of theinstant invention comprise DLL3 modulators in the form of antibodiesthat preferentially associate with one or more domains of an isoform ofDLL3 protein and, optionally, other DLL family members. Those ofordinary skill in the art will appreciate the well developed knowledgebase on antibodies such as set forth, for example, in Abbas et al.,Cellular and Molecular Immunology, 6^(th) ed., W. B. Saunders Company(2010) or Murphey et al., Janeway's Immunobiology, 8^(th) ed., GarlandScience (2011), each of which is incorporated herein by reference in itsentirety.

The term “antibody” is intended to cover polyclonal antibodies,muiticlonal antibodies, monoclonal antibodies, chimeric antibodies,humanized and primatized antibodies, human antibodies, recombinantlyproduced antibodies, intrabodies, multispecific antibodies, bispecificantibodies, monovalent antibodies, multivalent antibodies,anti-idiotypic antibodies, synthetic antibodies, including muteins andvariants thereof; antibody fragments such as Fab fragments, F(ab′)fragments, single-chain FvFvs, single-chain Fvs; and derivatives thereofincluding Fc fusions and other modifictaions, and any otherimmunologically active molecule so long as they exhibit the desiredbiological activity (i.e., antigen association or binding). Moreover,the term further comprises all classes of antibodies (i.e. IgA, IgD,IgE, IgG, and IgM) and all isotypes (i.e., IgG1, IgG2, IgG3, IgG4, IgA1,and IgA2), as well as variations thereof unless otherwise dictated bycontext. Heavy-chain constant domains that correspond to the differentclasses of antibodies are denoted by the corresponding lower case Greekletter α, δ, ε, γ, and μ, respectively. Light chains of the antibodiesfrom any vertebrate species can be assigned to one of two clearlydistinct types, called kappa (κ) and lambda (λ), based on the amino acidsequences of their constant domains.

While all such antibodies are within the scope of the present invention,preferred embodiments comprising the IgG class of immunoglobulin will bediscussed in some detail herein solely for the purposes of illustration.It will be understood that such disclosure is, however, merelydemonstrative of exemplary compositions and methods of practicing thepresent invention and not in any way limiting of the scope of theinvention or the claims appended hereto.

As is well known, the variable domains of both the light (V_(L)) andheavy (V_(H)) chain portions determine antigen recognition andspecificity and the constant domains of the light chain (C_(L)) and theheavy chain (C_(H)1, C_(H)2 or C_(H)3) confer and regulate importantbiological properties such as secretion, transplacental mobility,circulation half-life, complement binding, and the like.

The “variable” region includes hypervariable sites that manifestthemselves in three segments commonly termed complementarity determiningregions (CDRs), in both the light-chain and the heavy-chain variabledomains. The more highly conserved portions of variable domains flankingthe CDRs are termed framework regions (FRs). For example, in naturallyoccurring monomeric immunoglobulin G (IgG) antibodies, the six CDRspresent on each arm of the “Y” are short, non-contiguous sequences ofamino acids that are specifically positioned to form the antigen bindingsite as the antibody assumes its three dimensional configuration in anaqueous environment. Thus, each naturally occurring IgG antibodycomprises two identical binding sites proximal to the amino-terminus ofeach arm of the Y.

It will be appreciated that the position of CDRs can be readilyidentified by one of ordinary skill in the art using standardtechniques. Also familiar to those in the art is the numbering systemdescribed in Kabat et al. (1991, NIH Publication 91-3242, NationalTechnical Information Service, Springfield, Va.). In this regard Kabatet al. defined a numbering system for variable domain sequences that isapplicable to any antibody. One of ordinary skill in the art canunambiguously assign this system of “Kabat numbering” to any variabledomain sequence, without reliance on any experimental data beyond thesequence itself. Unless otherwise specified, references to the numberingof specific amino acid residue positions in an antibody are according tothe Kabat numbering system.

Thus, according to Kabat, in the V_(H), residues 31-35 comprise CDR1,residues 50-65 make up CDR2, and 95-102 comprise CDR3, while in theV_(L), residues 24-34 are CDR1, 50-56 comprise CDR2, and 89-97 make upCDR3. For context, in a V_(H), FR1 corresponds to the domain of thevariable region encompassing amino acids 1-30; FR2 corresponds to thedomain of the variable region encompassing amino acids 36-49; FR3corresponds to the domain of the variable region encompassing aminoacids 66-94, and FR4 corresponds to the domain of the variable regionfrom amino acids 103 to the end of the variable region. The FRs for thelight chain are similarly separated by each of the light chain variableregion CDRs.

Note that CDRs vary considerably from antibody to antibody (and bydefinition will not exhibit homology with the Kabat consensussequences). In addition, the identity of certain individual residues atany given Kabat site number may vary from antibody chain to antibodychain due to interspecies or allelic divergence. Alternative numberingis set forth in Chothia et al., J. Mol. Biol. 196:901-917 (1987) andMacCallum et at., J. Mol. Biol. 262:732-745 (1996), although as inKabat, the FR boundaries are separated by the respective CDR termini asdescribed above. See also Chothia et al., Nature 342, pp. 877-883 (1989)and S. Dubel, ed., Handbook of Therapeutic Antibodies, 3^(rd) ed.,WILEY-VCH Verlag GmbH and Co. (2007), where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Each of the aforementioned references is incorporated herein byreference in its entirety and the amino acid residues which comprisebinding regions or CDRs as defined by each of the above cited referencesand are set forth for comparison below.

CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-35 26-32 30-35V_(H) CDR2 50-65 50-58 47-58 V_(H) CDR3  95-102  95-102  93-101 V_(L)CDR1 24-34 23-34 30-36 V_(L) CDR2 50-56 50-56 46-55 V_(L) CDR3 89-9789-97 89-96 ¹Residue numbering follows the nomenclature of Kabat et al.,supra ²Residue numbering follows the nomenclature of Chothia et al.,supra ³Residue numbering follows the nomenclature of MacCallum et al.,supra

In the context of the instant invention it will be appreciated that anyof the disclosed light and heavy chain CDRs derived from the murinevariable region amino acid sequences set forth in FIG. 11A or FIG. 11Bmay be combined or rearranged to provide optimized anti-DLL3 (e.g.humanized or chimeric anti-hDLL3) antibodies in accordance with theinstant teachings. That is, one or more of the CDRs derived from thecontiguous light chain variable region amino acid sequences set forth inFIG. 11A (SEQ ID NOS: 20-202, even numbers) or the contiguous heavychain variable region amino acid sequences set forth in FIG. 11B (SEQ IDNOS: 21-203, odd numbers) may be incorporated in a DLL3 modulator and,in particularly preferred embodiments, in a CDR grafted or humanizedantibody that immunospecifically associates with one or more DLL3isoforms. Examples of light (SEQ ID NOS: 204-212, even numbers) andheavy (SEQ ID NOS: 205-213, odd numbers) chain variable region aminoacid sequences of such humanized modulators are also set forth in FIGS.11A and 11B. Taken together these novel amino acid sequences depictninety-two murine and five humanized exemplary modulators in accordancewith the instant invention. Moreover, corresponding nucleic acidsequences of each of the ninety-two exemplary murine modulators and fivehumanized modulators set forth in FIGS. 11A and 11B are included in thesequence listing appended to the instant application (SEQ ID NOS:220-413).

In FIGS. 11A and 11B the annotated CDRs are defined using Chothianumbering. However, as discussed herein and demonstrated in Example 8below, one skilled in the art could readily define, identify, deriveand/or enumerate the CDRs as defined by Kabat et al., Chothia et al. orMacCallum et al. for each respective heavy and light chain sequence setforth in FIG. 11A or FIG. 11B. Accordingly, each of the subject CDRs andantibodies comprising CDRs defined by all such nomenclature areexpressly included within the scope of the instant invention. Morebroadly, the terms “variable region CDR amino acid residue” or moresimply “CDR” includes amino acids in a CDR as identified using anysequence or structure based method as set forth above.

2. Antibody Modulator Generation

a. Polyclonal Antibodies

The production of polyclonal antibodies in various host animals,including rabbits, mice, rats, etc. is well known in the art. In someembodiments, polyclonal anti-DLL3 antibody-containing serum is obtainedby bleeding or sacrificing the animal. The serum may be used forresearch purposes in the form obtained from the animal or, in thealternative, the anti-DLL3 antibodies may be partially or fully purifiedto provide immunoglobulin fractions or homogeneous antibodypreparations.

Briefly the selected animal is immunized with a DLL3 immunogen (e.g.,soluble DLL3 or sDLL3) which may, for example, comprise selectedisoforms, domains and/or peptides, or live cells or cell preparationsexpressing DLL3 or immunoreactive fragments thereof. Art known adjuvantsthat may be used to increase the immunological response, depending onthe inoculated species include, but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants mayprotect the antigen from rapid dispersal by sequestering it in a localdeposit, or they may contain substances that stimulate the host tosecrete factors that are chemotactic for macrophages and othercomponents of the immune system. Preferably the immunization schedulewill involve two or more administrations of the selected immunogenspread out over a predetermined period of time.

The amino acid sequence of a DLL3 protein as shown in FIGS. 1C or 1D canbe analyzed to select specific regions of the DLL3 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of a DLL3 amino acid sequence are used to identify hydrophilicregions in the DLL3 structure. Regions of a DLL3 protein that showimmunogenic structure, as well as other regions and domains, can readilybe identified using various other methods known in the art, such asChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis. Average Flexibility profiles can be generatedusing the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept.Protein Res. 32:242-255. Beta-turn profiles can be generated using themethod of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.Thus, each DLL3 region, domain or motif identified by any of theseprograms or methods is within the scope of the present invention and maybe isolated or engineered to provide immunogens giving rise tomodulators comprising desired properties. Preferred methods for thegeneration of DLL3 antibodies are further illustrated by way of theExamples provided herein. Methods for preparing a protein or polypeptidefor use as an immunogen are well known in the art. Also well known inthe art are methods for preparing immunogenic conjugates of a proteinwith a carrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents are effective.Administration of a DLL3 immunogen is often conducted by injection overa suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken as described in the Examples below to determineadequacy of antibody formation.

b. Monoclonal Antibodies

In addition, the invention contemplates use of monoclonal antibodies. Asknown in the art, the term “monoclonal antibody” (or mAb) refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible mutations (e.g., naturally occurringmutations), that may be present in minor amounts. In certainembodiments, such a monoclonal antibody includes an antibody comprisinga polypeptide sequence that binds or associates with an antigen whereinthe antigen-binding polypeptide sequence was obtained by a process thatincludes the selection of a single target binding polypeptide sequencefrom a plurality of polypeptide sequences.

More generally, and as exemplified in Example 6 herein, monoclonalantibodies can be prepared using a wide variety of techniques known inthe art including hybridoma, recombinant techniques, phage displaytechnologies, transgenic animals (e.g., a XenoMouse®) or somecombination thereof. For example, monoclonal antibodies can be producedusing hybridoma and art-recognized biochemical and genetic engineeringtechniques such as described in more detail in An, Zhigiang (ed.)Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley andSons, 1^(st) ed. 2009; Shire et. al. (eds.) Current Trends in MonoclonalAntibody Development and Manufacturing, Springer Science+Business MediaLLC, 1^(st) ed. 2010; Harlow et at., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling, et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981) each of which is incorporated herein in its entirety by reference.It should be understood that a selected binding sequence can be furtheraltered, for example, to improve affinity for the target, to humanizethe target binding sequence, to improve its production in cell culture,to reduce its immunogenicity in vivo, to create a multispecificantibody, etc., and that an antibody comprising the altered targetbinding sequence is also an antibody of this invention.

c. Chimeric Antibodies

In another embodiment, the antibody of the invention may comprisechimeric antibodies derived from covalently joined protein segments fromat least two different species or types of antibodies. As known in theart, the term “chimeric” antibodies is directed to constructs in which aportion of the heavy and/or light chain is identical with or homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)).

In one embodiment, a chimeric antibody in accordance with the teachingsherein may comprise murine V_(H) and V_(L) amino acid sequences andconstant regions derived from human sources. In other compatibleembodiments a chimeric antibody of the present invention may comprise ahumanized antibody as described below. In another embodiment, theso-called “CDR-grafted” antibody, the antibody comprises one or moreCDRs from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the antibody chain(s) is/areidentical with or homologous to a corresponding sequence in antibodiesderived from another species or belonging to another antibody class orsubclass. For use in humans, selected rodent CDRs may be grafted into ahuman antibody, replacing one or more of the naturally occurringvariable regions or CDRs of the human antibody. These constructsgenerally have the advantages of providing full strength modulatorfunctions (e.g., CDC (complement dependent cytotoxicity), ADCC(antibody-dependent cell-mediated cytotoxicity), etc.) while reducingunwanted immune responses to the antibody by the subject.

d. Humanized Antibodies

Similar to the CDR-grafted antibody is a “humanized” antibody. As usedherein, “humanized” forms of non-human (e.g., murine) antibodies arechimeric antibodies that contain a minimal sequence derived from one ormore non-human immunoglobulins. In one embodiment, a humanized antibodyis a human immunoglobulin (recipient or acceptor antibody) in whichresidues from a CDR of the recipient are replaced by residues from a CDRof a non-human species (donor antibody) such as mouse, rat, rabbit, ornonhuman primate having the desired specificity, affinity, and/orcapacity. In certain preferred embodiments, residues in one or more FRsin the variable domain of the human immunoglobulin are replaced bycorresponding non-human residues from the donor antibody to helpmaintain the appropriate three-dimensional configuration of the graftedCDR(s) and thereby improve affinity. Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody to, for example, further refine antibody performance.

CDR grafting and humanized antibodies are described, for example, inU.S. Pat. Nos. 6,180,370 and 5,693,762. The humanized antibodyoptionally may also comprise at least a portion of an immunoglobulin Fc,typically that of a human immunoglobulin. For further details, see,e.g., Jones et al., Nature 321:522-525 (1986); and U.S. Pat. Nos.6,982,321 and 7,087,409. Still another method is termed “humaneering”which is described, for example, in U.S.P.N. 2005/0008625. Additionally,a non-human antibody may also be modified by specific deletion of humanT-cell epitopes or “deimmunization” by the methods disclosed in WO98/52976 and WO 00/34317. Each of the aforementioned references areincorporated herein in their entirety.

Humanized antibodies may also be bioengineered using common molecularbiology techniques, such as isolating, manipulating, and expressingnucleic acid sequences that encode all or part of immunoglobulinvariable regions from at least one of a heavy or light chain. Inaddition to the sources of such nucleic acid noted above, human germlinesequences are available as disclosed, for example, in Tomlinson, I. A.et al. (1992) J. Mol. Biol. 227:776-798; Cook, G. P. et al. (1995)Immunol. Today 16: 237-242; Chothia, D. et al. (1992) J. Mol. Biol.227:799-817; and Tomlinson et al. (1995) EMBO J 14:4628-4638. The V-BASEdirectory (VBASE2—Retter et al., Nucleic Acid Res. 33; 671-674, 2005)provides a comprehensive directory of human immunoglobulin variableregion sequences (compiled by Tomlinson, I. A. et al. MRC Centre forProtein Engineering, Cambridge, UK). Consensus human FRs can also beused, e.g., as described in U.S. Pat. No. 6,300,064.

In selected embodiments, and as detailed in Example 8 below, at least60%, 65%, 70%, 75%, or 80% of the humanized or CDR grafted antibodyheavy or light chain variable region amino acid residues will correspondto those of the recipient human FR and CDR sequences. In otherembodiments at least 85% or 90% of the humanized antibody variableregion residues will correspond to those of the recipient FR and CDRsequences. In a further preferred embodiment, greater than 95% of thehumanized antibody variable region residues will correspond to those ofthe recipient FR and CDR sequences.

e. Human Antibodies

In another embodiment, the antibodies may comprise fully humanantibodies. The term “human antibody” refers to an antibody whichpossesses an amino acid sequence that corresponds to that of an antibodyproduced by a human and/or has been made using any of the techniques formaking human antibodies.

Human antibodies can be produced using various techniques known in theart. One technique is phage display in which a library of (preferablyhuman) antibodies is synthesized on phages, the library is screened withthe antigen of interest or an antibody-binding portion thereof, and thephage that binds the antigen is isolated, from which one may obtain theimmunoreactive fragments. Methods for preparing and screening suchlibraries are well known in the art and kits for generating phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; and theStratagene SurfZAP™ phage display kit, catalog no. 240612). There alsoare other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., U.S. Pat. No.5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791,WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; and Barbas et al.,Proc. Natl. Acad. Sci. USA 88:7978-7982 (1991)).

In one embodiment, recombinant human antibodies may be isolated byscreening a recombinant combinatorial antibody library prepared asabove. In one embodiment, the library is a scFv phage display library,generated using human V_(L) and V_(H) cDNAs prepared from mRNA isolatedfrom B-cells.

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(a) of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in the art. Forexample, mutation can be introduced at random in vitro by usingerror-prone polymerase (reported in Leung et al., Technique, 1: 11-15(1989)). Additionally, affinity maturation can be performed by randomlymutating one or more CDRs, e.g. using PCR with primers carrying randomsequence spanning the CDR of interest, in selected individual Fv clonesand screening for higher-affinity clones. WO 9607754 described a methodfor inducing mutagenesis in a CDR of an immunoglobulin light chain tocreate a library of light chain genes. Another effective approach is torecombine the V_(H) or V_(L) domains selected by phage display withrepertoires of naturally occurring V domain variants obtained fromunimmunized donors and to screen for higher affinity in several roundsof chain reshuffling as described in Marks et al., Biotechnol., 10:779-783 (1992). This technique allows the production of antibodies andantibody fragments with a dissociation constant K_(D)(k_(off)/k_(on)) ofabout 10⁻⁹ M or less.

In other embodiments, similar procedures may be employed using librariescomprising eukaryotic cells (e.g., yeast) that express binding pairs ontheir surface. See, for example, U.S. Pat. No. 7,700,302 and U.S. Ser.No. 12/404,059. In one embodiment, the human antibody is selected from aphage library, where that phage library expresses human antibodies(Vaughan et al. Nature Biotechnology 14:309-314 (1996): Sheets et al.Proc. Natl. Acad. Sci. USA 95:6157-6162 (1998). In other embodiments,human binding pairs may be isolated from combinatorial antibodylibraries generated in eukaryotic cells such as yeast. See e.g., U.S.Pat. No. 7,700,302. Such techniques advantageously allow for thescreening of large numbers of candidate modulators and provide forrelatively easy manipulation of candidate sequences (e.g., by affinitymaturation or recombinant shuffling).

Human antibodies can also be made by introducing human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated andhuman immunoglobulin genes have been introduced. Upon challenge, humanantibody production is observed, which closely resembles that seen inhumans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXenoMouse® technology; and Lonberg and Huszar, Intern. Rev. Immunol.13:65-93 (1995). Alternatively, the human antibody may be prepared viaimmortalization of human B lymphocytes producing an antibody directedagainst a target antigen (such B lymphocytes may be recovered from anindividual suffering from a neoplastic disorder or may have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol,147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373.

3. Further Processing

No matter how obtained, modulator-producing cells (e.g., hybridomas,yeast colonies, etc.) may be selected, cloned and further screened fordesirable characteristics including, for example, robust growth, highantibody production and, as discussed in more detail below, desirableantibody characteristics. Hybridomas can be expanded in vivo insyngeneic animals, in animals that lack an immune system, e.g., nudemice, or in cell culture in vitro. Methods of selecting, cloning andexpanding hybridomas and/or colonies, each of which produces a discreteantibody species, are well known to those of ordinary skill in the art.

B. Recombinant: Modulator Production

1. Overview

Once the source is perfected DNA encoding the desired DLL3 modulatorsmay be readily isolated and sequenced using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding antibody heavy and light chains).Isolated and subcloned hybridoma cells (or phage or yeast derivedcolonies) may serve as a preferred source of such DNA if the modulatoris an antibody. If desired, the nucleic acid can further be manipulatedas described herein to create agents including fusion proteins, orchimeric, humanized or fully human antibodies. More particularly,isolated DNA (which may be modified) can be used to clone constant andvariable region sequences for the manufacture antibodies.

Accordingly, in exemplary embodiments antibodies may be producedrecombinantly, using conventional procedures (such as those set forth inAl-Rubeai; An, and Shire et. al. all supra, and Sambrook J. & Russell D.Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, Wiley, John & Sons, Inc. (2002)) inwhich the isolated and subcloned hybridoma cells (or phage or yeastderived colonies) serve as a preferred source of nucleic acid molecules.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules and artificial variants thereof (e.g.,peptide nucleic acids), whether single-stranded or double-stranded. Thenucleic acids may encode one or both chains of an antibody of theinvention, or a fragment or derivative thereof. The nucleic acidmolecules of the invention also include polynucleotides sufficient foruse as hybridization probes, PCR primers or sequencing primers foridentifying, analyzing, mutating or amplifying a polynucleotide encodinga polypeptide; anti-sense nucleic acids for inhibiting expression of apolynucleotide, and as well as complementary sequences. The nucleicacids can be any length. They can be, for example, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400,450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides in length,and/or can comprise one or more additional sequences, for example,regulatory sequences, and/or be part of a larger nucleic acid, forexample, a vector. It will be appreciated that such nucleic acidsequences can further be manipulated to create modulators includingchimeric, humanized or fully human antibodies. More particularly,isolated nucleic acid molecules (which may be modified) can be used toclone constant and variable region sequences for the manufactureantibodies as described in U.S. Pat. No. 7,709,611.

The term “isolated nucleic acid” means a that the nucleic acid was (i)amplified in vitro, for example by polymerase chain reaction (PCR), (ii)recombinantly produced by cloning, (iii) purified, for example bycleavage and gel-electrophoretic fractionation, or (iv) synthesized, forexample by chemical synthesis. An isolated nucleic acid is a nucleicacid that is available for manipulation by recombinant DNA techniques.

Whether the source of the nucleic acid encoding the desiredimmunoreactive portion of the antibody is obtained or derived from phagedisplay technology, yeast libraries, hybridoma-based technology orsynthetically, it is to be understood that the present inventionencompasses the nucleic acid molecules and sequences encoding theantibodies or antigen-binding fragments or derivatives thereof. Further,the instant invention is directed to vectors and host cells comprisingsuch nucleic acid molecules.

2. Hybridization and Sequence Identity

As indicated, the invention further provides nucleic acids thathybridize to other nucleic acids under particular hybridizationconditions. More specifically the invention encompasses nucleic acidsmolecules that hybridize under moderate or high stringency hybridizationconditions (e.g., as defined below), to the nucleic acid molecules ofthe invention. Methods for hybridizing nucleic acids are well-known inthe art. As is well known, a moderately stringent hybridizationconditions comprise a prewashing solution containing 5× sodiumchloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization buffer of about 50% formamide, 6×SSC, and a hybridizationtemperature of 55° C. (or other similar hybridization solutions, such asone containing about 50% formamide, with a hybridization temperature of42° C.), and washing conditions of 60° C., in 0.5×SSC, 0.1% SDS. By wayof comparison hybridization under highly stringent hybridizationconditions comprise washing with 6×SSC at 45° C., followed by one ormore washes in 0.1×SSC, 0.2% SDS at 68° C. Furthermore, one of skill inthe art can manipulate the hybridization and/or washing conditions toincrease or decrease the stringency of hybridization such that nucleicacids comprising nucleotide sequences that are at least 65%, 70%, 75%,80%, 85%, 90%, 95%, 98% or 99% identical to each other typically remainhybridized to each other.

The invention also includes nucleic acid molecules that are“substantially identical” to the described nucleic acid molecules. Inone embodiment, the term substantially identical with regard to anucleic acid sequence means may be construed as a sequence of nucleicacid molecules exhibiting at least about 65%, 70%, 75%, 80%, 85%, or 90%sequence identity. In other embodiments, the nucleic acid moleculesexhibit 95% or 98% sequence identity to the reference nucleic acidsequence.

The basic parameters affecting the choice of hybridization conditionsand guidance for devising suitable conditions are set forth by, forexample, Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., chapters 9 and 11; and Current Protocols in MolecularBiology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections2.10 and 6.3-6.4), and can be readily determined by those havingordinary skill in the art based on, for example, the length and/or basecomposition of the nucleic acid.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, the sequence analysis tool GCG (Accelrys Software Inc.)contains programs such as “GAP” and “BEST-FIT” which can be used withdefault parameters to determine sequence homology or sequence identitybetween closely related polypeptides, such as homologous polypeptidesfrom different species of organisms or between a wild type protein and amutein thereof. (See, e.g., GCG Version 6.1 or Durbin et. Al.,Biological Sequence Analysis: Probabilistic models of proteins andnucleic acids., Cambridge Press (1998)).

Polypeptide sequences can also be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially blastp or tblastn, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215: 403 410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389 402, each of which is hereinincorporated by reference.

In this regard the invention also includes nucleic acid molecules thatencode polypeptides that are “substantially identical” with respect toan antibody variable region polypeptide sequence (e.g., either the donorlight or heavy chain variable region or the acceptor light or heavychain variable region). As applied to such polypeptides, the term“substantial identity” or “substantially identical” means that twopeptide sequences, when optimally aligned, such as by the programs GAPor BEST-FIT using default gap weights, share at least 60% or 65%sequence identity, preferably at least 70%, 75%, 80%, 85%, or 90%sequence identity, even more preferably at least 93%, 95%, 98% or 99%sequence identity. Preferably, residue positions which are not identicaldiffer by conservative amino acid substitutions. A “conservative aminoacid substitution” is one in which an amino acid residue is substitutedby another amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of similarity may be adjustedupwards to correct for the conservative nature of the substitution.

3. Expression

The varied processes of recombinant expression, i.e., the production ofRNA or of RNA and protein/peptide, are well known as set forth, forexample, in Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego,Calif.; Sambrook et al., Molecular Cloning-A Laboratory Manual (3rdEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,(2000); and Current Protocols in Molecular Biology, F. M. Ausubel etal., eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (supplemented through2006).

Certain terms of interest include “expression control sequence” whichcomprises promoters, ribosome binding sites, enhancers and other controlelements which regulate transcription of a gene or translation of mRNA.As is well known, a “promoter” or “promoter region” relates to a nucleicacid sequence which generally is located upstream (5′) to the nucleicacid sequence being expressed and controls expression of the sequence byproviding a recognition and binding site for RNA-polymerase.

Exemplary promoters which are compatible according to the inventioninclude promoters for SP6, T3 and T7 polymerase, human U6 RNA promoter,CMV promoter, and artificial hybrid promoters thereof (e.g. CMV) where apart or parts are fused to a part or parts of promoters of genes ofother cellular proteins such as e.g. human GAPDH(glyceraldehyde-3-phosphate dehydrogenase), and including or notincluding (an) additional intron(s).

In certain embodiments, the nucleic acid molecule may be present in avector, where appropriate with a promoter, which controls expression ofthe nucleic acid. The well known term “vector” comprises anyintermediary vehicle for a nucleic acid which enables said nucleic acid,for example, to be introduced into prokaryotic and/or eukaryotic cellsand, where appropriate, to be integrated into a genome. Methods oftransforming mammalian cells are well known in the art. See, forexample, U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455.The vectors may include a nucleotide sequence encoding an antibody ofthe invention (e.g., a whole antibody, a heavy or light chain of anantibody, a V_(H) or V_(L) of an antibody, or a portion thereof, or aheavy- or light-chain CDR, a single chain Fv, or fragments or variantsthereof), operably linked to a promoter (see, e.g., PCT Publication WO86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464).

A variety of host-expression vector systems are commercially available,and many are compatible with the teachings herein and may be used toexpress the modulators of the invention. Such systems include, but arenot limited to, microorganisms such as bacteria (e.g., E. coli, B.subtilis, streptomyces) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing modulator codingsequences; yeast (e.g., Saccharomyces, Pichia) transfected withrecombinant yeast expression vectors containing modulator codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing modulator codingsequences; plant cell systems (e.g., Nicotiana, Arabidopsis, duckweed,corn, wheat, potato, etc.) infected with recombinant viral expressionvectors (e.g., cauliflower mosaic virus; tobacco mosaic virus) ortransfected with recombinant plasmid expression vectors (e.g., Tiplasmid) containing modulator coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells, etc.) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

As used herein, the term “host cell” covers any kind of cellular systemwhich can be engineered to generate the polypeptides and antigen-bindingmolecules of the present invention. In one embodiment, the host cell isengineered to allow the production of an antigen binding molecule withmodified glycoforms. In a preferred embodiment, the antigen bindingmolecule, or variant antigen binding molecule, is an antibody, antibodyfragment, or fusion protein. In certain embodiments, the host cells havebeen further manipulated to express increased levels of one or morepolypeptides having N-acetylglucosaminyltransferase III (GnTIII)activity. Compatible host cells include cultured cells, e.g., mammaliancultured cells, such as CHO cells, BHK cells, NSO cells, SP2/0 cells, YOmyeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells orhybridoma cells, yeast cells, insect cells, and plant cells, to nameonly a few, but also cells comprised within a transgenic animal,transgenic plant or cultured plant or animal tissue.

For long-term, high-yield production of recombinant proteins stableexpression is preferred. Accordingly, cell lines that stably express theselected modulator may be engineered using standard art-recognizedtechniques. Rather than using expression vectors that contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Any of the selection systems well knownin the art may be used, including the glutamine synthetase geneexpression system (the GS system) which provides an efficient approachfor enhancing expression under certain conditions. The GS system isdiscussed in whole or part in connection with EP patents 0 216 846, 0256 055, 0 323 997 and 0 338 841 and U.S. Pat. Nos. 5,591,639 and5,879,936 each of which is incorporated herein by reference. Anotherpreferred expression system, the Freedom™ CHO-S Kit is commerciallyprovided by Life Technologies (Catalog Number A13696-01) also allows forthe development of stable cell lines that may be used for modulatorproduction.

Such host-expression systems represent vehicles by which the codingsequences of interest may be produced and subsequently purified, butalso represent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express a molecule of theinvention in situ. The host cell may be co-transfected with twoexpression vectors of the invention, for example, the first vectorencoding a heavy chain derived polypeptide and the second vectorencoding a light chain derived polypeptide.

Thus, in certain embodiments, the present invention provides recombinanthost cells allowing for the expression of antibodies or portionsthereof. Antibodies produced by expression in such recombinant hostcells are referred to herein as recombinant antibodies. The presentinvention also provides progeny cells of such host cells, and antibodiesproduced by the same.

C. Chemical Synthesis

In addition, the modulators may be chemically synthesized usingtechniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller, M., et al., 1984, Nature 310:105-111). Furthermore, ifdesired, nonclassical amino acids or chemical amino acid analogs (suchas D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, and the like) can be introduced asa substitution or addition into a polypeptide sequence.

D. Transgenic Systems

In other embodiments modulators may be produced transgenically throughthe generation of a mammal or plant that is transgenic for recombinantmolecules such as the immunoglobulin heavy and light chain sequences andthat produces the desired compounds in a recoverable form. Thisincludes, for example, the production of protein modulators (e.g.,antibodies) in, and recovery from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957. In some embodiments, non-human transgenic animals thatcomprise human immunoglobulin loci are immunized to produce antibodies.

Other transgenic techniques are set forth in Hogan et al., Manipulatingthe Mouse Embryo: A Laboratory Manual 2nd ed., Cold Spring Harbor Press(1999); Jackson et al., Mouse Genetics and Transgenics: A PracticalApproach, Oxford University Press (2000); and Pinkert, Transgenic AnimalTechnology: A Laboratory Handbook, Academic Press (1999) and U.S. Pat.No. 6,417,429. In some embodiments, the non-human animals are mice,rats, sheep, pigs, goats, cattle or horses, and the desired product isproduced in blood, milk, urine, saliva, tears, mucus and other bodilyfluids from which it is readily obtainable using art-recognizedpurification techniques.

Other compatible production systems include methods for makingantibodies in plants such as described, for example, in U.S. Pat. Nos.6,046,037 and 5,959,177 which are incorporated herein with respect tosuch techniques.

E. Isolation/Purification

Once a modulator of the invention has been produced by recombinantexpression or any other of the disclosed techniques, it may be purifiedby any method known in the art for purification of immunoglobulins orproteins. In this respect the modulator may be “isolated” which meansthat it has been identified and separated and/or recovered from acomponent of its natural environment. Contaminant components of itsnatural environment are materials that would interfere with diagnosticor therapeutic uses for the polypeptide and may include enzymes,hormones, and other proteinaceous or nonproteinaceous solutes. Isolatedmodulators include a modulator in situ within recombinant cells becauseat least one component of the polypeptides natural environment will notbe present.

If the desired molecule is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, may beremoved, for example, by centrifugation or ultrafiltration. Where themodulator is secreted into the medium, supernatants from such expressionsystems are generally first concentrated using a commercially availableprotein concentration filter, for example, an Amicon or Pelliconultrafiltration unit (Millipore Corp.). Once the insoluble contaminantsare removed the modulator preparation may be further purified usingstandard techniques such as, for example, hydroxylapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography of particular interest. Inthis regard protein A can be used to purify antibodies that are based onhuman IgG1, IgG2 or IgG4 heavy chains (Lindmark, et al., J Immunol Meth62:1 (1983)) while protein G is recommended for all mouse isotypes andfor human IgG3 (Guss, et al., EMBO J 5:1567 (1986)). Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, reverse phase HPLC, chromatography onsilica, chromatography on heparin, sepharose chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered. In particularlypreferred embodiments the modulators of the instant invention will bepurified, at least in part, using Protein A or Protein G affinitychromatography.

VI. DLL3 Modulator Fragments and Derivatives

Whatever generation and production methodology is selected, modulatorsof the instant invention will react, bind, combine, complex, connect,attach, join, interact or otherwise associate with a target determinant(e.g., antigen) and thereby provide the desired results. Where themodulator comprises an antibody or fragment, construct or derivativethereof such associations may be through one or more “binding sites” or“binding components” expressed on the antibody, where a binding sitecomprises a region of a polypeptide that is responsible for selectivelybinding to a target molecule or antigen of interest. Binding domainscomprise at least one binding site (e.g., an intact IgG antibody willhave two binding domains and two binding sites). Exemplary bindingdomains include an antibody variable domain, a receptor-binding domainof a ligand, a ligand-binding domain of a receptor or an enzymaticdomain.

A. Antibodies

As noted above, the term “antibody” is intended to cover, at least,polyclonal antibodies, multiclonal antibodies, chimeric antibodies, CDRgrafted antibodies, humanized and primatized antibodies, humanantibodies, recombinantly produced antibodies, intrabodies,multispecific antibodies, bispecific antibodies, monovalent antibodies,multivalent antibodies, anti-idiotypic antibodies, as well as syntheticantibodies.

B. Fragments

Regardless of which form of the modulator (e.g. chimeric, humanized,etc.) is selected to practice the invention it will be appreciated thatimmunoreactive fragments of the same may be used in accordance with theteachings herein. An “antibody fragment” comprises at least a portion ofan intact antibody. As used herein, the term “fragment” of an antibodymolecule includes antigen-binding fragments of antibodies, and the term“antigen-binding fragment” refers to a polypeptide fragment of animmunoglobulin or antibody that immunospecifically binds or reacts witha selected antigen or immunogenic determinant thereof or competes withthe intact antibody from which the fragments were derived for specificantigen binding.

Exemplary fragments include: V_(L), V_(H), scFv, F(ab′)2 fragment, Fabfragment, Fd fragment, Fv fragment, single domain antibody fragments,diabodies, linear antibodies, single-chain antibody molecules andmultispecific antibodies formed from antibody fragments. In addition, anactive fragment comprises a portion of the antibody that retains itsability to interact with the antigen/substrates or receptors and modifythem in a manner similar to that of an intact antibody (though maybewith somewhat less efficiency).

In other embodiments, an antibody fragment is one that comprises the Fcregion and that retains at least one of the biological functionsnormally associated with the Fc region when present in an intactantibody, such as FcRn binding, antibody half-life modulation, ADCCfunction and complement binding. In one embodiment, an antibody fragmentis a monovalent antibody that has an in vivo half-life substantiallysimilar to an intact antibody. For example, such an antibody fragmentmay comprise an antigen binding arm linked to an Fc sequence capable ofconferring in vivo stability to the fragment.

As would be well recognized by those skilled in the art, fragments canbe obtained via chemical or enzymatic treatment (such as papain orpepsin) of an intact or complete antibody or antibody chain or byrecombinant means. See, e.g., Fundamental Immunology, W. E. Paul, ed.,Raven Press, N.Y. (1999), for a more detailed description of antibodyfragments.

C. Derivatives

The invention further includes immunoreactive modulator derivatives andantigen binding molecules comprising one or more modifications.

1. Multivalent Antibodies

In one embodiment, the modulators of the invention may be monovalent ormultivalent (e.g., bivalent, trivalent, etc.). As used herein, the term“valency” refers to the number of potential target binding sitesassociated with an antibody. Each target binding site specifically bindsone target molecule or specific position or locus on a target molecule.When an antibody is monovalent, each binding site of the molecule willspecifically bind to a single antigen position or epitope. When anantibody comprises more than one target binding site (multivalent), eachtarget binding site may specifically bind the same or differentmolecules (e.g., may bind to different ligands or different antigens, ordifferent epitopes or positions on the same antigen). See, for example,U.S.P.N. 2009/0130105. In each case at least one of the binding siteswill comprise an epitope, motif or domain associated with a DLL3isoform.

In one embodiment, the modulators are bispecific antibodies in which thetwo chains have different specificities, as described in Millstein etal., 1983, Nature, 305:537-539. Other embodiments include antibodieswith additional specificities such as trispecific antibodies. Other moresophisticated compatible multispecific constructs and methods of theirfabrication are set forth in U.S.P.N. 2009/0155255, as well as WO94/04690; Suresh et al., 1986, Methods in Enzymology, 121:210; andWO96/27011.

As alluded to above, multivalent antibodies may immunospecifically bindto different epitopes of the desired target molecule or mayimmunospecifically bind to both the target molecule as well as aheterologous epitope, such as a heterologous polypeptide or solidsupport material. While preferred embodiments of the anti-DLL3antibodies only bind two antigens (i.e. bispecific antibodies),antibodies with additional specificities such as trispecific antibodiesare also encompassed by the instant invention. Bispecific antibodiesalso include cross-linked or “heteroconjugate” antibodies. For example,one of the antibodies in the heteroconjugate can be coupled to avidin,the other to biotin. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP03089). Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

In yet other embodiments, antibody variable domains with the desiredbinding specificities (antibody-antigen combining sites) are fused toimmunoglobulin constant domain sequences, such as an immunoglobulinheavy chain constant domain comprising at least part of the hinge,C_(H)2, and/or C_(H)3 regions, using methods well known to those ofordinary skill in the art.

2. Fc Region Modifications

In addition to the various modifications, substitutions, additions ordeletions to the variable or binding region of the disclosed modulators(e.g., Fc-DLL3 or anti-DLL3 antibodies) set forth above, those skilledin the art will appreciate that selected embodiments of the presentinvention may also comprise substitutions or modifications of theconstant region (i.e. the Fc region). More particularly, it iscontemplated that the DLL3 modulators of the invention may contain interalia one or more additional amino acid residue substitutions, mutationsand/or modifications which result in a compound with preferredcharacteristics including, but not limited to: altered pharmacokinetics,increased serum half life, increase binding affinity, reducedimmunogenicity, increased production, altered Fc ligand binding to an Fcreceptor (FcR), enhanced or reduced “ADCC” (antibody-dependent cellmediated cytotoxicity) or “CDC” (complement-dependent cytotoxicity)activity, altered glycosylation and/or disulfide bonds and modifiedbinding specificity. In this regard it will be appreciated that these Fcvariants may advantageously be used to enhance the effectiveanti-neoplastic properties of the disclosed modulators.

To this end certain embodiments of the invention may comprisesubstitutions or modifications of the Fc region, for example theaddition of one or more amino acid residue, substitutions, mutationsand/or modifications to produce a compound with enhanced or preferred Fceffector functions. For example, changes in amino acid residues involvedin the interaction between the Fc domain and an Fc receptor (e.g.,FcγRI, FcγRIIA and B, FcγRIII and FcRn) may lead to increasedcytotoxicity and/or altered pharmacokinetics, such as increased serumhalf-life (see, for example, Ravetch and Kinet, Annu. Rev. Immunol9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haaset al., J. Lab. Clin. Med. 126:330-41 (1995) each of which isincorporated herein by reference).

In selected embodiments, antibodies with increased in vivo half-livescan be generated by modifying (e.g., substituting, deleting or adding)amino acid residues identified as involved in the interaction betweenthe Fc domain and the FcRn receptor (see, e.g., InternationalPublication Nos. WO 97/34631; WO 04/029207; U.S. Pat. No. 6,737,056 andU.S.P.N. 2003/0190311. With regard to such embodiments, Fc variants mayprovide half-lives in a mammal, preferably a human, of greater than 5days, greater than 10 days, greater than 15 days, preferably greaterthan 20 days, greater than 25 days, greater than 30 days, greater than35 days, greater than 40 days, greater than 45 days, greater than 2months, greater than 3 months, greater than 4 months, or greater than 5months. The increased half-life results in a higher serum titer whichthus reduces the frequency of the administration of the antibodiesand/or reduces the concentration of the antibodies to be administered.Binding to human FcRn in vivo and serum half life of human FcRn highaffinity binding polypeptides can be assayed, e.g., in transgenic miceor transfected human cell lines expressing human FcRn, or in primates towhich the polypeptides with a variant Fc region are administered. WO2000/42072 describes antibody variants with improved or diminishedbinding to FcRns. See also, e.g., Shields et al. J. Biol. Chem.9(2):6591-6604 (2001).

In other embodiments, Fc alterations may lead to enhanced or reducedADCC or CDC activity. As in known in the art, CDC refers to the lysingof a target cell in the presence of complement, and ADCC refers to aform of cytotoxicity in which secreted Ig bound onto FcRs present oncertain cytotoxic cells (e.g., Natural Killer cells, neutrophils, andmacrophages) enables these cytotoxic effector cells to bind specificallyto an antigen-bearing target cell and subsequently kill the target cellwith cytotoxins. In the context of the instant invention antibodyvariants are provided with “altered” FcR binding affinity, which iseither enhanced or diminished binding as compared to a parent orunmodified antibody or to an antibody comprising a native sequence FcR.Such variants which display decreased binding may possess little or noappreciable binding, e.g., 0-20% binding to the FcR compared to a nativesequence, e.g. as determined by techniques well known in the art. Inother embodiments the variant will exhibit enhanced binding as comparedto the native immunoglobulin Fc domain. It will be appreciated thatthese types of Fc variants may advantageously be used to enhance theeffective anti-neoplastic properties of the disclosed antibodies. In yetother embodiments, such alterations lead to increased binding affinity,reduced immnunogenicity, increased production, altered glycosylationand/or disulfide bonds (e.g., for conjugation sites), modified bindingspecificity, increased phagocytosis; and/or down regulation of cellsurface receptors (e.g. B cell receptor; BCR), etc.

3. Altered Glycosylation

Still other embodiments comprise one or more engineered glycoforms,i.e., a DLL3 modulator comprising an altered glycosylation pattern oraltered carbohydrate composition that is covalently attached to theprotein (e.g., in the Fc domain). See, for example, Shields, R. L. etal. (2002) J. Biol. Chem. 277:26733-26740. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function, increasing the affinity of the modulatorfor a target or facilitating production of the modulator. In certainembodiments where reduced effector function is desired, the molecule maybe engineered to express an aglycosylated form. Substitutions that mayresult in elimination of one or more variable region frameworkglycosylation sites to thereby eliminate glycosylation at that site arewell known (see e.g. U.S. Pat. Nos. 5,714,350 and 6,350,861).Conversely, enhanced effector functions or improved binding may beimparted to the Fc containing molecule by engineering in one or moreadditional glycosylation sites.

Other embodiments include an Fc variant that has an alteredglycosylation composition, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNAc structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Engineeredglycoforms may be generated by any method known to one skilled in theart, for example by using engineered or variant expression strains, byco-expression with one or more enzymes (for exampleN-acetylglucosaminyltransferase III (GnTIII)), by expressing a moleculecomprising an Fc region in various organisms or cell lines from variousorganisms or by modifying carbohydrate(s) after the molecule comprisingFc region has been expressed (see, for example, WO 2012/117002).

4. Additional Processing

The modulators may be differentially modified during or afterproduction, e.g., by glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications may be carried outby known techniques, including but not limited, to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄, acetylation, formylation, oxidation, reduction,metabolic synthesis in the presence of tunicamycin, etc.

Various post-translational modifications also encompassed by theinvention include, for example, N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends, attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. Moreover, the modulators may also be modified with adetectable label, such as an enzymatic, fluorescent, radioisotopic oraffinity label to allow for detection and isolation of the modulator.

VII. Modulator Characteristics

No matter how obtained or which of the aforementioned forms themodulator takes, various embodiments of the disclosed modulators mayexhibit certain characteristics. In selected embodiments,antibody-producing cells (e.g., hybridomas or yeast colonies) may beselected, cloned and further screened for favorable propertiesincluding, for example, robust growth, high modulator production and, asdiscussed in more detail below, desirable modulator characteristics. Inother cases characteristics of the modulator may be imparted orinfluenced by selecting a particular antigen (e.g., a specific DLL3isoform) or immunoreactive fragment of the target antigen forinoculation of the animal. In still other embodiments the selectedmodulators may be engineered as described above to enhance or refineimmunochemical characteristics such as affinity or pharmacokinetics.

A. Neutralizina Modulators

In certain embodiments, the modulators will comprise “neutralizing”antibodies or derivatives or fragments thereof. That is, the presentinvention may comprise antibody molecules that bind specific domains,motifs or epitopes and are capable of blocking, reducing or inhibitingthe biological activity of DLL3. More generally the term “neutralizingantibody” refers to an antibody that binds to or interacts with a targetmolecule or ligand and prevents binding or association of the targetmolecule to a binding partner such as a receptor or substrate, therebyinterrupting a biological response that otherwise would result from theinteraction of the molecules.

It will be appreciated that competitive binding assays known in the artmay be used to assess the binding and specificity of an antibody orimmunologically functional fragment or derivative thereof. With regardto the instant invention an antibody or fragment will be held to inhibitor reduce binding of DLL3 to a binding partner or substrate when anexcess of antibody reduces the quantity of binding partner bound to DLL3by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%,99% or more as measured, for example, by Notch receptor activity or inan in vitro competitive binding assay. In the case of antibodies to DLL3for example, a neutralizing antibody or antagonist will preferably alterNotch receptor activity by at least about 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 95%, 97%, 99% or more. It will be appreciated that thismodified activity may be measured directly using art-recognizedtechniques or may be measured by the impact the altered activity hasdownstream (e.g., oncogenesis, cell survival or activation orsuppression of Notch responsive genes). Preferably, the ability of anantibody to neutralize DLL3 activity is assessed by inhibition of DLL3binding to a Notch receptor or by assessing its ability to relieve DLL3mediated repression of Notch signaling.

B. Internalizing Modulators

There is evidence that a substantial portion of expressed DLL3 proteinremains associated with the tumorigenic cell surface, thereby allowingfor localization and internalization of the disclosed modulators. Inpreferred embodiments such modulators may be associated with, orconjugated to, anti-cancer agents such as cytotoxic moieties that killthe cell upon internalization. In particularly preferred embodiments themodulator will comprise an internalizing antibody drug conjugate.

As used herein, a modulator that “internalizes” is one that is taken up(along with any payload) by the cell upon binding to an associatedantigen or receptor. As will be appreciated, the internalizing modulatormay, in preferred embodiments, comprise an antibody including antibodyfragments and derivatives thereof, as well as antibody conjugates.Internalization may occur in vitro or in vivo. For therapeuticapplications, internalization will preferably occur in vivo in a subjectin need thereof. The number of antibody molecules internalized may besufficient or adequate to kill an antigen-expressing cell, especially anantigen-expressing cancer stem cell. Depending on the potency of theantibody or antibody conjugate, in some instances, the uptake of asingle antibody molecule into the cell is sufficient to kill the targetcell to which the antibody binds. For example, certain toxins are sohighly potent that the internalization of a few molecules of the toxinconjugated to the antibody is sufficient to kill the tumor cell. Whetheran antibody internalizes upon binding to a mammalian cell can bedetermined by various assays including those described in the Examplesbelow (e.g., Examples 12 and 15-17). Methods of detecting whether anantibody internalizes into a cell are also described in U.S. Pat. No.7,619,068 which is incorporated herein by reference in its entirety.

C. Depleting Modulators

In other embodiments the antibodies will comprise depleting antibodiesor derivatives or fragments thereof. The term “depleting” antibodyrefers to an antibody that preferably binds to or associates with anantigen on or near the cell surface and induces, promotes or causes thedeath or elimination of the cell (e.g., by CDC, ADCC or introduction ofa cytotoxic agent). In some embodiments, the selected depletingantibodies will be associated or conjugated to a cytotoxic agent.

Preferably a depleting antibody will be able to remove, incapacitate,eliminate or kill at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,95%, 97%, or 99% of DLL3 tumorigenic cells in a defined cell population.In some embodiments the cell population may comprise enriched,sectioned, purified or isolated tumor perpetuating cells. In otherembodiments the cell population may comprise whole tumor samples orheterogeneous tumor extracts that comprise tumor perpetuating cells.Those skilled in the art will appreciate that standard biochemicaltechniques as described in the Examples below (e.g., Examples 13 and 14)may be used to monitor and quantify the depletion of tumorigenic cellsor tumor perpetuating cells in accordance with the teachings herein.

D. Binning and Epitope Binding

It will further be appreciated the disclosed anti-DLL3 antibodymodulators will associate with, or bind to, discrete epitopes orimmunogenic determinants presented by the selected target or fragmentthereof. In certain embodiments, epitope or immunogenic determinantsinclude chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, incertain embodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. Thus, as usedherein the term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor or otherwiseinteracting with a molecule. In certain embodiments, an antibody is saidto specifically bind (or immunospecifically bind or react) an antigenwhen it preferentially recognizes its target antigen in a complexmixture of proteins and/or macromolecules. In preferred embodiments, anantibody is said to specifically bind an antigen when the equilibriumdissociation constant (K_(D)) is less than or equal to 10⁻⁶M or lessthan or equal to 10⁻⁷M, more preferably when the equilibriumdissociation constant is less than or equal to 10⁻⁸M, and even morepreferably when the dissociation constant is less than or equal to 10⁻⁹M

More directly the term “epitope” is used in its common biochemical senseand refers to that portion of the target antigen capable of beingrecognized and specifically bound by a particular antibody modulator.When the antigen is a polypeptide such as DLL3, epitopes may generallybe formed from both contiguous amino acids and noncontiguous amino acidsjuxtaposed by tertiary folding of a protein (“conformational epitopes”).In such conformational epitopes the points of interaction occur acrossamino acid residues on the protein that are linearly separated from oneanother. Epitopes formed from contiguous amino acids (sometimes referredto as “linear” or “continuous” epitopes) are typically retained uponprotein denaturing, whereas epitopes formed by tertiary folding aretypically lost upon protein denaturing. In any event an antibody epitopetypically includes at least 3, and more usually, at least 5 or 8-10amino acids in a unique spatial conformation.

In this respect it will be appreciated that, in certain embodiments, anepitope may be associated with, or reside in, one or more regions,domains or motifs of the DLL3 protein (e.g., amino acids 1-618 ofisoform 1). As discussed in more detail herein the extracellular regionof the DLL3 protein comprises a series of generally recognized domainsincluding six EGF-like domains and a DSL domain. For the purposes of theinstant disclosure the term “domain” will be used in accordance with itsgenerally accepted meaning and will be held to refer to an identifiableor definable conserved structural entity within a protein that exhibitsa distinctive secondary structure content. In many cases, homologousdomains with common functions will usually show sequence similaritiesand be found in a number of disparate proteins (e.g., EGF-like domainsare reportedly found in at least 471 different proteins). Similarly, theart-recognized term “motif” will be used in accordance with its commonmeaning and shall generally refer to a short, conserved region of aprotein that is typically ten to twenty contiguous amino acid residues.As discussed throughout, selected embodiments comprise modulators thatassociate with or bind to an epitope within specific regions, domains ormotifs of DLL3.

In any event once a desired epitope on an antigen is determined, it ispossible to generate antibodies to that epitope, e.g., by immunizingwith a peptide comprising the epitope using techniques described in thepresent invention. Alternatively, during the discovery process, thegeneration and characterization of antibodies may elucidate informationabout desirable epitopes located in specific domains or motifs. Fromthis information, it is then possible to competitively screen antibodiesfor binding to the same epitope. An approach to achieve this is toconduct competition studies to find antibodies that competitively bindwith one another, i.e. the antibodies compete for binding to theantigen. A high throughput process for binning antibodies based upontheir cross-competition is described in WO 03/48731. Other methods ofbinning or domain level or epitope mapping comprising modulatorcompetition or antigen fragment expression on yeast is set forth inExamples 9 and 10 below.

As used herein, the term “binning” refers to methods used to group orclassify antibodies based on their antigen binding characteristics andcompetition. While the techniques are useful for defining andcategorizing modulators of the instant invention, the bins do not alwaysdirectly correlate with epitopes and such initial determinations ofepitope binding may be further refined and confirmed by otherart-recognized methodology as described herein. However, as discussedand shown in the Examples below, empirical assignment of antibodymodulators to individual bins provides information that may beindicative of the therapeutic potential of the disclosed modulators.

More specifically, one can determine whether a selected referenceantibody (or fragment thereof) binds to the same epitope or crosscompetes for binding with a second test antibody (i.e., is in the samebin) by using methods known in the art and set forth in the Examplesherein. In one embodiment, a reference antibody modulator is associatedwith DLL3 antigen under saturating conditions and then the ability of asecondary or test antibody modulator to bind to DLL3 is determined usingstandard immunochemical techniques. If the test antibody is able tosubstantially bind to DLL3 at the same time as the reference anti-DLL3antibody, then the secondary or test antibody binds to a differentepitope than the primary or reference antibody. However, if the testantibody is not able to substantially bind to DLL3 at the same time,then the test antibody binds to the same epitope, an overlappingepitope, or an epitope that is in close proximity (at least sterically)to the epitope bound by the primary antibody. That is, the test antibodycompetes for antigen binding and is in the same bin as the referenceantibody.

The term “compete” or “competing antibody” when used in the context ofthe disclosed modulators means competition between antibodies asdetermined by an assay in which a test antibody or immunologicallyfunctional fragment under test prevents or inhibits specific binding ofa reference antibody to a common antigen. Typically, such an assayinvolves the use of purified antigen (e.g., DLL3 or a domain or fragmentthereof) bound to a solid surface or cells bearing either of these, anunlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excessand/or allowed to bind first. Antibodies identified by competition assay(competing antibodies) include antibodies binding to the same epitope asthe reference antibody and antibodies binding to an adjacent epitopesufficiently proximal to the epitope bound by the reference antibody forsteric hindrance to occur. Additional details regarding methods fordetermining competitive binding are provided in the Examples herein.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least30%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, bindingis inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

Conversely, when the reference antibody is bound it will preferablyinhibit binding of a subsequently added test antibody (i.e., a DLL3modulator) by at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. Insome instance, binding of the test antibody is inhibited by at least80%, 85%, 90%, 95%, or 97% or more.

With regard to the instant invention, and as set forth in the Examples 9and 10 below, it has been determined (via surface plasmon resonance orbio-layer interferometry) that the extracellular domain of DLL3 definesat least nine bins by competitive binding termed “bin A” to “bin I”herein. Given the resolution provided by modulator binning techniques,it is believed that these nine bins comprise the majority of the binsthat are present in the extracellular region of the DLL3 protein.

In this respect, and as known in the art and detailed in the Examplesbelow, the desired binning or competitive binding data can be obtainedusing solid phase direct or indirect radioimmunoassay (RIA), solid phasedirect or indirect enzyme immunoassay (EIA or ELISA), sandwichcompetition assay, it Biacore™ 2000 system (i.e,, surface plasmonresonance—GE Healthcare), a ForteBio® Analyzer (i.e., bio-layerinterferometry—ForteBio, Inc.) or flow cytometric methodology. The term“surface plasmon resonance,” as used herein, refers to an opticalphenomenon that allows for the analysis of real-time specificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix. The term “bio-layer interferometry” refers toan optical analytical technique that analyzes the interference patternof white light reflected from two surfaces: a layer of immobilizedprotein on a biosensor tip, and an internal reference layer. Any changein the number of molecules bound to the biosensor tip causes a shift inthe interference pattern that can be measured in real-time. Inparticularly preferred embodiments the analysis (whether surface plasmonresonance, bio-layer interferometry or flow cytometry) is performedusing a Biacore or ForteBio instrument or a flow cytometer (e.g.,FACSAria II) as demonstrated in the Examples below.

In order to further characterize the epitopes that the disclosed DLL3antibody modulators associate with or bind to, domain-level epitopemapping was performed using a modification of the protocol described byCochran et al. (J Immunol Methods. 287 (1-2):147-158 (2004) which isincorporated herein by reference). Briefly, individual domains of DLL3comprising specific amino acid sequences were expressed on the surfaceof yeast and binding by each DLL3 antibody was determined through flowcytometry. The results are discussed below in Example 10 and shown inFIGS. 14A and 14B.

Other compatible epitope mapping techniques include alanine scanningmutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63)(herein specifically incorporated by reference in its entirety), orpeptide cleavage analysis. In addition, methods such as epitopeexcision, epitope extraction and chemical modification of antigens canbe employed (Tomer (2000) Protein Science 9: 487-496) (hereinspecifically incorporated by reference in its entirety). In otherembodiments Modification-Assisted Profiling (MAP), also known as AntigenStructurebased Antibody Profiling (ASAP) provides a method thatcategorizes large numbers of monoclonal antibodies (mAbs) directedagainst the same antigen according to the similarities of the bindingprofile of each antibody to chemically or enzymatically modified antigensurfaces (U.S.P.N. 2004/0101920, herein specifically incorporated byreference in its entirety). Each category may reflect a unique epitopeeither distinctly different from or partially overlapping with epitoperepresented by another category. This technology allows rapid filteringof genetically identical antibodies, such that characterization can befocused on genetically distinct antibodies. It will be appreciated thatMAP may be used to sort the hDLL3 antibody modulators of the inventioninto groups of antibodies binding different epitopes

Agents useful for altering the structure of the immobilized antigeninclude enzymes such as proteolytic enzymes (e.g., trypsin,endoproteinase Glu-C, endoproteinase Asp-N, chymotrypsin, etc.). Agentsuseful for altering the structure of the immobilized antigen may also bechemical agents, such as, succinimidyl esters and their derivatives,primary amine-containing compounds, hydrazines and carbohydrazines, freeamino acids, etc.

The antigen protein may be immobilized on either biosensor chip surfacesor polystyrene beads. The latter can be processed with, for example, anassay such as multiplex LUMINEX™ detection assay (Luminex Corp.).Because of the capacity of LUMINEX to handle multiplex analysis with upto 100 different types of beads, LUMINEX provides almost unlimitedantigen surfaces with various modifications, resulting in improvedresolution in antibody epitope profiling over a biosensor assay.

E. Modulator Binding Characteristics

Besides epitope specificity the disclosed antibodies may becharacterized using physical characteristics such as, for example,binding affinities. In this regard the present invention furtherencompasses the use of antibodies that have a high binding affinity forone or more DLL3 isoforms or, in the case of pan-antibodies, more thanone member of the DLL family.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction. Anantibody of the invention is said to immunospecifically bind its targetantigen when the dissociation constant K_(D)(k_(off)/k_(on)) is ≦10⁻⁷M.The antibody specifically binds antigen with high affinity when theK_(D) is ≦5×10⁻⁹M, and with very high affinity when the K_(D) is≦5×10⁻¹⁰M. In one embodiment of the invention, the antibody has a K_(D)of ≦10⁻⁹M and an off-rate of about 1×10⁻⁴/sec. In one embodiment of theinvention, the off-rate is <1×10⁻⁵/sec. In other embodiments of theinvention, the antibodies will bind to DLL3 with a K_(D) of betweenabout 10⁻⁷M and 10⁻¹⁰M, and in yet another embodiment it will bind witha K_(D) ≦2×10⁻¹⁰M. Still other selected embodiments of the presentinvention comprise antibodies that have a disassociation constant orK_(D)(k_(off)/k_(on)) of less than 10⁻²M, less than 5×10⁻²M, less than10⁻³M, less than 5×10⁻³M, less than 10⁻⁴M, less than 5×10⁻⁴M, less than10⁻⁵M, less than 5×10⁻⁵M, less than 10⁻⁶M, less than 5×10⁻⁶M, less than10⁻⁷M, less than 5×10⁻⁷M, less than 10⁻⁸M, less than 5×10⁻⁸M, less than10⁻⁹M, less than 5×10⁻⁹M, less than 10 ⁻¹⁰M, less than 5×10⁻¹⁰M, lessthan 10⁻¹¹M, less than 5×10⁻¹¹M, less than 10⁻¹²M, less than 5×10⁻¹²M,less than 10⁻¹³M, less than 5×10⁻¹³M, less than 10⁻¹⁴M, less than5×10⁻¹⁴M, less than 10⁻¹⁵M or less than 5×10⁻¹⁵M.

In specific embodiments, an antibody of the invention thatimmunospecifically binds to DLL3 has an association rate constant ork_(on) (or k_(a)) rate (DLL3 (Ab)+antigen (Ag)^(k) _(on)←Ab-Ag) of atleast 10⁵M⁻¹s⁻¹, at least 2×10⁵M⁻¹s⁻¹, at least 5×10⁵M⁻¹s⁻¹, at least10⁶M⁻¹s⁻¹, at least 5×10⁶M⁻¹s⁻¹, at least 10⁷M⁻¹s⁻¹, at least5×10⁷M⁻¹s⁻¹, or at least 10⁸M⁻¹s⁻¹.

In another embodiment, an antibody of the invention thatimmunospecifieally binds to DLL3 has a disassociation rate constant ork_(off) (or k_(d)) rate (DLL3 (Ab)+antigen (Ag)^(k) _(off)←Ab-Ag) ofless than 10⁻¹s⁻¹, less than 5×10⁻¹s⁻¹, less than 10⁻²s⁻¹, less than5×10⁻²s⁻¹, less than 10⁻³s⁻¹, less than 5×10⁻³s⁻¹, less than 10⁻⁴s⁻¹,less than 5×10⁻⁴s⁻¹, less than 10⁻⁵s⁻¹, less than 5×10⁻⁵s⁻¹, less than10⁻⁶s⁻¹, less than 5×10⁻⁶s⁻¹, less than 10⁻⁷s⁻¹, less than 5×10⁻⁷s⁻¹,less than 10⁻⁸s⁻¹, less than 5×10⁻⁸s⁻¹, less than 10⁻⁹s⁻¹, less than5×10⁻⁹s⁻¹ or less than 10⁻¹⁰s⁻¹.

In other selected embodiments of the present invention anti-DLL3antibodies will have an affinity constant or k_(a) (k_(on)/k_(off)) ofat least 10²M⁻¹, at least 5×10²M⁻¹, at least 10³M⁻¹, at least 5×10³M⁻¹,at least 10⁴M⁻¹, at least 5×10⁴M⁻¹, at least 10⁵M⁻¹, at least 5×10⁵M⁻¹,at least 10⁶M⁻¹, at least 5×10⁶M⁻¹, at least 10⁷M⁻¹, at least 5×10⁷M⁻¹,at least 10⁸M⁻¹, at least 5×10⁸M⁻¹, at least 10⁹M⁻¹, at least 5×10⁹M⁻¹,at least 10¹⁰M⁻¹, at least 5×10¹⁰M⁻¹, at least 10¹¹M⁻¹, at least5×10¹¹M⁻¹, at least 10¹²M⁻¹, at least 5×10¹²M⁻¹, at least 10¹³M⁻¹, atleast 5×10¹³M⁻¹, at least 10¹⁴M⁻¹, at least 5×10¹⁴M⁻¹, at least 10¹⁵M⁻¹or at least 5×10¹⁵M⁻¹.

Besides the aforementioned modulator characteristics antibodies of theinstant invention may further be characterized using additional physicalcharacteristics including, for example, thermal stability (i.e, meltingtemperature; Tm), and isoelectric points. (See, e.g., Bjellqvist et al.,1993, Electrophoresis 14:1023; Vermeer et al., 2000, Biophys. J.78:394-404; Vermeer et al., 2000, Biophys. J. 79: 2150-2154 each ofwhich is incorporated herein by reference).

VIII. Conjugated Modulators

A. Overview

Once the modulators of the invention have been generated and/orfabricated and selected according to the teachings herein they may belinked with, fused to, conjugated to (e.g., covalently ornon-covalently) or otherwise associated with pharmaceutically active ordiagnostic moieties or biocompatible modifiers. As used herein the term“conjugate” or “modulator conjugate” or “antibody conjugate” will beused broadly and held to mean any biologically active or detectablemolecule or drug associated with the disclosed modulators regardless ofthe method of association. In this respect it will be understood thatsuch conjugates may, in addition to the disclosed modulators, comprisepeptides, polypeptides, proteins, prodrugs which are metabolized to anactive agent in vivo, polymers, nucleic acid molecules, small molecules,binding agents, mimetic agents, synthetic drugs, inorganic molecules,organic molecules and radioisotopes. Moreover, as indicated above theselected conjugate may be covalently or non-covalently associated with,or linked to, the modulator and exhibit various stoichiometric molarratios depending, at least in part, on the method used to effect theconjugation.

Particularly preferred aspects of the instant invention will compriseantibody modulator conjugates or antibody-drug conjugates that may beused for the diagnosis and/or treatment of proliferative disorders. Itwill be appreciated that, unless otherwise dictated by context, the term“antibody-drug conjugate” or “ADC” or the formula M-[L-D]n shall be heldto encompass conjugates comprising both therapeutic and diagnosticmoieties. In such embodiments antibody-drug conjugate compounds willcomprise a DLL3 modulator (typically an anti-DLL3 antibody) as themodulator or cellular binding unit (abbreviated as CBA, M, or Abherein), a therapeutic (e.g., anti-cancer agent) or diagnostic moiety(D), and optionally a linker (L) that joins the drug and the antigenbinding agent. For the purposes of the instant disclosure “n” shall beheld to mean an integer from 1 to 20. In a preferred embodiment, themodulator is a DLL3 mAb comprising at least one CDR from the heavy andlight chain variable regions as described above.

Those skilled in the art will appreciate that a number of differentreactions are available for the attachment or association of therapeuticor diagnostic moieties and/or linkers to binding agents. In selectedembodiments this may be accomplished by reaction of the amino acidresidues of the binding agent, e.g., antibody molecule, including theamine groups of lysine, the free carboxylic acid groups of glutamic andaspartic acid, the sulfhydryl groups of cysteine and the variousmoieties of the aromatic amino acids. One of the most commonly usednon-specific methods of covalent attachment is the carbodiimide reactionto link a carboxy (or amino) group of a compound to amino (or carboxy)groups of the antibody. Additionally, bifunctional agents such asdialdehydes or imidoesters have been used to link the amino group of acompound to amino groups of an antibody molecule. Also available forattachment of drugs to binding agents is the Schiff base reaction. Thismethod involves the periodate oxidation of a drug that contains glycolor hydroxy groups, thus forming an aldehyde which is then reacted withthe binding agent. Attachment occurs via formation of a Schiff base withamino groups of the binding agent. Isothiocyanates and azlactones canalso be used as coupling agents for covalently attaching drugs tobinding agents.

In other embodiments the disclosed modulators of the invention may beconjugated or associated with proteins, polypeptides or peptides thatimpart selected characteristics (e.g., biotoxins, biomarkers,purification tags, etc.). In certain preferred embodiments the presentinvention encompasses the use of modulators or fragments thereofrecombinantly fused or chemically conjugated (including both covalentand non-covalent conjugations) to a heterologous protein or peptidewherein the protein or peptide comprises at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids. The construct does notnecessarily need to be directly linked, but may occur through amino acidlinker sequences. For example, antibodies may be used to targetheterologous polypeptides to particular cell types expressing DLL3,either in vitro or in vivo, by fusing or conjugating the modulators ofthe present invention to antibodies specific for particular cell surfacereceptors to provide bispecific constructs. Moreover, modulators fusedor conjugated to heterologous polypeptides may also be used in in vitroimmunoassays and may be particularly compatible with purificationmethodology (e.g., his-tags) as is known in the art. See e.g.,International publication No. WO 93/21232; European Patent No. EP439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat. No.5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et al.,1991, J. Immunol. 146:2446-2452.

B. Linkers

Besides the aforementioned peptide linkers or spacers, it will beappreciated that several other varieties or types of linker may be usedto associate the disclosed modulators with pharmaceutically active ordiagnostic moieties or biocompatible modifiers. In some embodiments, thelinker is cleavable under intracellular conditions, such that cleavageof the linker releases the drug unit from the antibody in theintracellular environment. In yet other embodiments, the linker unit isnot cleavable and the drug is released, for example, by antibodydegradation.

The linkers of the ADC are preferably stable extracellularly, preventaggregation of ADC molecules and keep the ADC freely soluble in aqueousmedia and in a monomeric state. Before transport or delivery into acell, the antibody-drug conjugate (ADC) is preferably stable and remainsintact, i.e. the antibody remains linked to the drug moiety. The linkersare stable outside the target cell and may be cleaved at someefficacious rate inside the cell. An effective linker will: (i) maintainthe specific binding properties of the antibody; (ii) allowintracellular delivery of the conjugate or drug moiety; (iii) remainstable and intact, i.e. not cleaved, until the conjugate has beendelivered or transported to its targeted site; and (iv) maintain acytotoxic, cell-killing effect or a cytostatic effect of the PBD drugmoiety. Stability of the ADC may be measured by standard analyticaltechniques such as mass spectroscopy, HPLC, and the separation/analysistechnique LC/MS. Covalent attachment of the antibody and the drug moietyrequires the linker to have two reactive functional groups, i.e.bivalency in a reactive sense. Bivalent linker reagents which are usefulto attach two or more functional or biologically active moieties, suchas peptides, nucleic acids, drugs, toxins, antibodies, haptens, andreporter groups are known, and methods have been described theirresulting conjugates (Hermanson, G. T. (1996) Bioconjugate Techniques;Academic Press: New York, p 234-242).

To this end certain embodiments of the invention comprise the use alinker that is cleavable by a cleaving agent that is present in theintracellular environment (e.g., within a lysosome or endosome orcaveolae). The linker can be, for example, a peptidyl linker that iscleaved by an intracellular peptidase or protease enzyme, including, butnot limited to, a lysosomal or endosomal protease. In some embodiments,the peptidyl linker is at least two amino acids long or at least threeamino acids long. Cleaving agents can include cathepsins B and D andplasmin, each of which is known to hydrolyze dipeptide drug derivativesresulting in the release of active drug inside target cells. Exemplarypeptidyl linkers that are cleavable by the thiol-dependent proteaseCathepsin-B are peptides comprising Phe-Leu since Cathepsin-B has beenfound to be highly expressed in cancerous tissue. Other examples of suchlinkers are described, for example, in U.S. Pat. No. 6,214,345 andU.S.P.N. 2012/0078028 each of which incorporated herein by reference inits entirety. In a specific preferred embodiment, the peptidyl linkercleavable by an intracellular protease is a Val-Cit linker, an Ala-Vallinker or a Phe-Lys linker such as is described in U.S. Pat. No.6,214,345. One advantage of using intracellular proteolytic release ofthe therapeutic agent is that the agent is typically attenuated whenconjugated and the serum stabilities of the conjugates are typicallyhigh.

In other embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (e.g., ahydrazone, oxime, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) can be used (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929). Such linkers are relativelystable under neutral pH conditions, such as those in the blood, but areunstable at below pH 5.5 or 5.0, the approximate pH of the lysosome.

In yet other embodiments, the linker is cleavable under reducingconditions (e.g., a disulfide linker). A variety of disulfide linkersare known in the art, including, for example, those that can be formedusing SATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio) butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).In yet other specific embodiments, the linker is a malonate linker(Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyllinker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a3′-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12). Inyet other embodiments, the linker unit is not cleavable and the drug isreleased by antibody degradation. (See U.S. Publication No. 2005/0238649incorporated by reference herein in its entirety and for all purposes).

More particularly, in preferred embodiments (set forth in U.S.P.N.2011/0256157 which is incorporated herein by reference in its entirety)compatible linkers will comprise:

where the asterisk indicates the point of attachment to the cytotoxicagent, CBA is a cell binding agent/modulator, L¹ is a linker, A is aconnecting group connecting L¹ to the cell binding agent, L² is acovalent bond or together with —OC(═O)— forms a self-immolative linker,and L¹ or L² is a cleavable linker.

L¹ is preferably the cleavable linker, and may be referred to as atrigger for activation of the linker for cleavage.

The nature of L¹ and L², where present, can vary widely. These groupsare chosen on the basis of their cleavage characteristics, which may bedictated by the conditions at the site to which the conjugate isdelivered. Those linkers that are cleaved by the action of enzymes arepreferred, although linkers that are cleavable by changes in pH (e.g.acid or base labile), temperature or upon irradiation (e.g. photolabile)may also be used. Linkers that are cleavable under reducing or oxidisingconditions may also find use in the present invention.

L¹ may comprise a contiguous sequence of amino acids. The amino acidsequence may be the target substrate for enzymatic cleavage, therebyallowing release of R¹⁰ from the N10 position.

In one embodiment, L¹ is cleavable by the action of an enzyme. In oneembodiment, the enzyme is an esterase or a peptidase.

In one embodiment, L² is present and together with —C(═O)O— forms aself-immolative linker. In one embodiment, L² is a substrate forenzymatic activity, thereby allowing release of R¹⁰ from the N10position.

In one embodiment, where L¹ is cleavable by the action of an enzyme andL² is present, the enzyme cleaves the bond between L¹ and L².

L¹ and L², where present, may be connected by a bond selected from:

—C(═O)NH—, —C(═O)O—, —NHC(═O)—, —OC(═O)—, —OC(═O)O—, —NHC(═O)O—,—OC(═O)NH—, and —NHC(═O)NH—.

An amino group of L¹ that connects to L² may be the N-terminus of anamino acid or may be derived from an amino group of an amino acid sidechain, for example a lysine amino acid side chain.

A carboxyl group of L¹ that connects to L² may be the C-terminus of anamino acid or may be derived from a carboxyl group of an amino acid sidechain, for example a glutamic acid amino acid side chain.

A hydroxyl group of L¹ that connects to L² may be derived from ahydroxyl group of an amino acid side chain, for example a serine aminoacid side chain.

The term “amino acid side chain” includes those groups found in: (i)naturally occurring amino acids such as alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine; (ii) minor amino acids suchas ornithine and citrulline; (iii) unnatural amino acids, beta-aminoacids, synthetic analogs and derivatives of naturally occurring aminoacids; and (iv) all enantiomers, diastereomers, isomerically enriched,isotopically labelled (e.g. ²H, ³H, ¹⁴C, ¹⁵N), protected forms, andracemic mixtures thereof.

In one embodiment, —C(═O)O— and L² together form the group:

where the asterisk indicates the point of attachment to the drug orcytotoxic agent position, the wavy line indicates the point ofattachment to the linker L¹, Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—,and a is 0 to 3. The phenylene ring is optionally substituted with one,two or three substituents as described herein. In one embodiment, thephenylene group is optionally substituted with halo, NO₂, R or OR.

In one embodiment, Y is NH.

In one embodiment, n is 0 or 1. Preferably, n is 0.

Where Y is NH and n is 0, the self-immolative linker may be referred toas a p-aminobenzylcarbonyl linker (PABC).

The self-immolative linker will allow for release of the protectedcompound when a remote site is activated, proceeding along the linesshown below (for n=0):

where L* is the activated form of the remaining portion of the linker.These groups have the advantage of separating the site of activationfrom the compound being protected. As described above, the phenylenegroup may be optionally substituted.

In one embodiment described herein, the group L* is a linker L¹ asdescribed herein, which may include a dipeptide group.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

where the asterisk, the wavy line, Y, and n are as defined above. Eachphenylene ring is optionally substituted with one, two or threesubstituents as described herein. In one embodiment, the phenylene ringhaving the Y substituent is optionally substituted and the phenylenering not having the Y substituent is unsubstituted. In one embodiment,the phenylene ring having the Y substituent is unsubstituted and thephenylene ring not having the Y substituent is optionally substituted.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

where the asterisk, the wavy line, Y, and n are as defined above, E isO, S or NR, D is N, CH, or CR, and F is N, CH, or CR.

In one embodiment, D is N.

In one embodiment, D is CH.

In one embodiment, E is O or S.

In one embodiment, F is CH.

In a preferred embodiment, the linker is a cathepsin labile linker.

In one embodiment, L¹ comprises a dipeptide. The dipeptide may berepresented as —NH—X₁—X₂—CO—, where —NH— and —CO— represent the N- andC-terminals of the amino acid groups X₁ and X₂ respectively. The aminoacids in the dipeptide may be any combination of natural amino acids.Where the linker is a cathepsin labile linker, the dipeptide may be thesite of action for cathepsin-mediated cleavage.

Additionally, for those amino acids groups having carboxyl or amino sidechain functionality, for example Glu and Lys respectively, CO and NH mayrepresent that side chain functionality.

In one embodiment, the group —X₁-X₂— in dipeptide, —NH—X₁-X₂—CO—, isselected from:

-Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-,-Leu-Cit-, -Ile-Cit-, -Phe-Arg- and -Trp-Cit- where Cit is citrulline.

Preferably, the group —X₁-X₂— in dipeptide, —NH—X₁-X₂—CO—, is selectedfrom:

-Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, and -Val-Cit-.

Most preferably, the group —X₁-X₂— in dipeptide, —NH—X₁-X₂—CO—, is-Phe-Lys- or -Val-Ala-.

Other dipeptide combinations may be used, including those described byDubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which isincorporated herein by reference.

In one embodiment, the amino acid side chain is derivatised, whereappropriate. For example, an amino group or carboxy group of an aminoacid side chain may be derivatised.

In one embodiment, an amino group NH₂ of a side chain amino acid, suchas lysine, is a derivatised form selected from the group consisting ofNHR and NRR′.

In one embodiment, a carboxy group COOH of a side chain amino acid, suchas aspartic acid, is a derivatised form selected from the groupconsisting of COOR, CONH₂, CONHR and CONRR′.

In one embodiment, the amino acid side chain is chemically protected,where appropriate. The side chain protecting group may be a group asdiscussed below in relation to the group R^(L). Protected amino acidsequences are cleavable by enzymes. For example, it has been establishedthat a dipeptide sequence comprising a Boc side chain-protected Lysresidue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known inthe art and are described in the Novabiochem Catalog. Additionalprotecting group strategies are set out in Protective Groups in OrganicSynthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those aminoacids having reactive side chain functionality:

Arg: Z, Mtr, Tos;

Asn: Trt, Xan;

Asp: Bzl, t-Bu;

Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;

Glu: Bzl, t-Bu;

Gln: Trt, Xan;

His: Boc, Dnp, Tos, Trt;

Lys: Boc, Z-Cl, Fmoc, Z, Alloc;

Ser: Bzl, TBDMS, TBDPS;

Thr: Bz;

Trp: Boc;

Tyr: Bzl, Z, Z-Br.

In one embodiment, the side chain protection is selected to beorthogonal to a group provided as, or as part of, a capping group, wherepresent. Thus, the removal of the side chain protecting group does notremove the capping group, or any protecting group functionality that ispart of the capping group.

In other embodiments of the invention, the amino acids selected arethose having no reactive side chain functionality. For example, theamino acids may be selected from: Ala, Gly, Ile, Leu, Met, Phe, Pro, andVal.

In one embodiment, the dipeptide is used in combination with aself-immolative linker. The self-immolative linker may be connected to—X₂—.

Where a self-immolative linker is present, —X₂— is connected directly tothe self-immolative linker. Preferably the group —X₂—CO— is connected toY, where Y is NH, thereby forming the group —X₂—CO—NH—.

—NH—X₁— is connected directly to A. A may comprise the functionality—CO— thereby to form an amide link with —X₁—.

In one embodiment, L¹ and L² together with —OC(═O)— comprise the groupNH—X₁-X₂—CO-PABC-. The PABC group is connected directly to the cytotoxicagent. Preferably, the self-immolative linker and the dipeptide togetherform the group —NH-Phe-Lys-CO—NH-PABC-, which is illustrated below:

where the asterisk indicates the point of attachment to the selectedcytotoxic moiety, and the wavy line indicates the point of attachment tothe remaining portion of the linker L¹ or the point of attachment to A.Preferably, the wavy line indicates the point of attachment to A. Theside chain of the Lys amino acid may be protected, for example, withBoc, Fmoc, or Alloc, as described above.

Alternatively, the self-immolative linker and the dipeptide togetherform the group —NH-Val-Ala-CO—NH-PABC-, which is illustrated below:

where the asterisk and the wavy line are as defined above.

Alternatively, the self-immolative linker and the dipeptide togetherform the group —NH-Val-Cit-CO—NH-PABC-, which is illustrated below:

where the asterisk and the wavy line are as defined above.

In some embodiments of the present invention, it may be preferred thatif the drug moiety contains an unprotected imine bond, e.g. if moiety Bis present, then the linker does not contain a free amino (H₂N—) group.Thus if the linker has the structure -A-L¹-L²- then this wouldpreferably not contain a free amino group. This preference isparticularly relevant when the linker contains a dipeptide, for exampleas L¹; in this embodiment, it would be preferred that one of the twoamino acids is not selected from lysine.

Without wishing to be bound by theory, the combination of an unprotectedimine bond in the drug moiety and a free amino group in the linker cancause dimerisation of the drug-linker moiety which may interfere withthe conjugation of such a drug-linker moiety to an antibody. Thecross-reaction of these groups may be accelerated in the case the freeamino group is present as an ammonium ion (H₃N⁺—), such as when a strongacid (e.g. TFA) has been used to deprotect the free amino group.

In one embodiment, A is a covalent bond. Thus, L¹ and the cell bindingagent are directly connected. For example, where L¹ comprises acontiguous amino acid sequence, the N-terminus of the sequence mayconnect directly to the cell binding agent.

Thus, where A is a covalent bond, the connection between the cellbinding agent and L¹ may be selected from:

—C(═O)NH—, —C(═O)O—, —NHC(═O)—, —OC(═O)—, —OC(═O)O—, —NHC(═O)O—,—OC(═O)NH—, —NHC(═O)NH—, —C(═O)NHC(═O)—, —S—, —S—S—, —CH₂C(═O)—, and═N—NH—.

An amino group of L¹ that connects to the DLL3 modulator may be theN-terminus of an amino acid or may be derived from an amino group of anamino acid side chain, for example a lysine amino acid side chain.

A carboxyl group of L¹ that connects to the modulator may be theC-terminus of an amino acid or may be derived from a carboxyl group ofan amino acid side chain, for example a glutamic acid amino acid sidechain.

A hydroxyl group of L¹ that connects to the cell binding agent may bederived from a hydroxyl group of an amino acid side chain, for example aserine amino acid side chain.

A thiol group of L¹ that connects to a modulator agent may be derivedfrom a thiol group of an amino acid side chain, for example a serineamino acid side chain.

The comments above in relation to the amino, carboxyl, hydroxyl andthiol groups of L¹ also apply to the cell binding agent.

In one embodiment, L² together with —OC(═O)— represents:

where the asterisk indicates the point of attachment to the N10position, the wavy line indicates the point of attachment to L¹, n is 0to 3, Y is a covalent bond or a functional group, and E is anactivatable group, for example by enzymatic action or light, thereby togenerate a self-immolative unit. The phenylene ring is optionallyfurther substituted with one, two or three substituents as describedherein. In one embodiment, the phenylene group is optionally furthersubstituted with halo, NO₂, R or OR. Preferably n is 0 or 1, mostpreferably 0.

E is selected such that the group is susceptible to activation, e.g. bylight or by the action of an enzyme. E may be —NO₂ or glucoronic acid.The former may be susceptible to the action of a nitroreductase, thelatter to the action of a β-glucoronidase.

In this embodiment, the self-immolative linker will allow for release ofthe protected compound when E is activated, proceeding along the linesshown below (for n=0):

where the asterisk indicates the point of attachment to the N10position, E* is the activated form of E, and Y is as described above.These groups have the advantage of separating the site of activationfrom the compound being protected. As described above, the phenylenegroup may be optionally further substituted.

The group Y may be a covalent bond to L¹.

The group Y may be a functional group selected from:

—C(═O)—, —NH—, —O—, —C(═O)NH—, —C(═O)O—, —NHC(═O)—, —OC(═O)—, —OC(═O)O—,—NHC(═O)O—, —OC(═O)NH—, —NHC(═O)NH—, —NHC(═O)NH, —C(═O)NHC(═O)—, and—S—.

Where L¹ is a dipeptide, it is preferred that Y is —NH— or —C(═O)—,thereby to form an amide bond between L¹ and Y. In this embodiment, thedipeptide sequence need not be a substrate for an enzymatic activity.

In another embodiment, A is a spacer group. Thus, L¹ and the cellbinding agent are indirectly connected.

L¹ and A may he connected by a bond selected from:

—C(═O)NH—, —C(═O)O—, —NHC(═O)—, —OC(═O)O—, —NHC(═O)O—, —OC(═O)NH—, and—NHC(═O)NH—.

Preferably, the linker contains an eleetrophilic functional group forreaction with a nucleophilic functional group on the modulator.Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) maleimide groups (ii) activated disulfides,(iii) active esters such as NHS (N-hydroxysuccinimide) esters, HOBt(N-hydroxybenzotriazole) esters, haloformates, and acid halides; (iv)alkyl and benzyl halides such as haloacetamides; and (v) aldehydes,ketones, carboxyl, and, some of which are exemplified as follows:

Certain antibodies have reducible interchain disulfides, i.e. cysteinebridges. Antibodies may be made reactive for conjugation with linkerreagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues). U.S. Pat. No. 7,521,541teaches engineering antibodies by introduction of reactive cysteineamino acids.

In some embodiments, a linker has a reactive nucleophilic group which isreactive with an electrophilic group present on an antibody. Usefulelectrophilic groups on an antibody include, but are not limited to,aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilicgroup of a Linker can react with an electrophilic group on an antibodyand form a covalent bond to an antibody unit. Useful nucleophilic groupson a linker include, but are not limited to, hydrazide, oxime, amino,hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide. The electrophilic group on an antibody provides aconvenient site for attachment to a Linker.

In one embodiment, the group A is:

where the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the cell binding agent, and nis 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A is:

where the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the cell binding agent, and nis 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A is:

where the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the cell binding agent, n is 0or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8. In anotherembodiment, m is 10 to 30, and preferably 20 to 30. Alternatively, m is0 to 50. In this embodiment, m is preferably 10-40 and n is 1.

In one embodiment, the group A is:

where the asterisk indicates the point of attachment to L¹, the wavyline indicates the point of attachment to the cell binding agent, n is 0or 1, and m is 0 to 30. In a preferred embodiment, n is 1 and m is 0 to10, 1 to 8, preferably 4 to 8, and most preferably 4 or 8. In anotherembodiment, m is 10 to 30, and preferably 20 to 30. Alternatively, m is0 to 50. In this embodiment, m is preferably 10-40 and n is 1.

In one embodiment, the connection between the cell binding agent and Ais through a thiol residue of the cell binding agent and a maleimidegroup of A.

In one embodiment, the connection between the cell binding agent and Ais:

where the asterisk indicates the point of attachment to the remainingportion of A and the wavy line indicates the point of attachment to theremaining portion of the cell binding agent. In this embodiment, the Satom is typically derived from the modulator.

In each of the embodiments above, an alternative functionality may beused in place of the maleimide-derived group shown below:

where the wavy line indicates the point of attachment to the cellbinding agent as before, and the asterisk indicates the bond to theremaining portion of the A group.

In one embodiment, the maleimide-derived group is replaced with thegroup:

where the wavy line indicates point of attachment to the cell bindingagent, and the asterisk indicates the bond to the remaining portion ofthe A group.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with the cell binding agent, is selected from:

—C(═O)NH—, —C(═O)O—, —NHC(═O)—, —OC(═O)—, —OC(═O)O—, —NHC(═O)O—,—OC(═O)NH—, —NHC(═O)NH—, —NHC(═O)NH, —C(═O)NHC(═O)—, —S—, —S—S—,—CH₂C(═O)—, —C(═O)CH₂—, ═N—NH— and —NH—N═.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with the cell binding agent, is selected from:

where the wavy line indicates either the point of attachment to the cellbinding agent or the bond to the remaining portion of the A group, andthe asterisk indicates the other of the point of attachment to the cellbinding agent or the bond to the remaining portion of the A group.

Other groups suitable for connecting L¹ to the selected modulator aredescribed in WO 2005/082023.

In another preferred embodiment the modulators of the instant inventionmay be associated with biocompatible polymers comprising drug linkerunits. In this respect one such type of compatible polymer comprisesFleximer® polymers (Mersana Therapeutics). Such polymers are reportedlybiodegradable, well tolerated and have been clinically validated.Moreover, such polymers are compatible with a number of customizablelinker technologies and chemistries allowing for control ofpharmacokinetics, localization of drug release and improvedbiodistribution.

The selected modulators can also be directly conjugated radioisotopes ormay comprise macrocyclic chelators useful for conjugating radiometalions (as described herein). In certain embodiments, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid(DOTA) which can be attached to the antibody via a linker molecule. Suchlinker molecules are commonly known in the art and described in Denardoet al., 1998, Clin Cancer Res. 4:2483; Peterson et al., 1999, Bioconjug.Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943.

More generally, techniques for conjugating therapeutic moieties orcytotoxic agents to modulators are well known. As discussed abovemoieties can be conjugated to modulators by any art-recognized method,including, but not limited to aldehyde/Schiff linkage, sulphydryllinkage, acid-labile linkage, cis-aconityl linkage, hydrazone linkage,enzymatically degradable linkage (see generally Garnett, 2002, Adv DrugDeliv Rev 53:171). Also see, e.g., Amon et al., “Monoclonal AntibodiesFor Immunotargeting Of Drugs In Cancer Therapy”, in MonoclonalAntibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (AlanR. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”,in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84; Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 301-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119. In preferred embodiments a DLL3 modulator that isconjugated to a therapeutic moiety or cytotoxic agent may beinternalized by a cell upon binding to a DLL3 molecule associated withthe cell surface thereby delivering the therapeutic payload.

C. Biocompatible Modifiers

In selected embodiments the modulators of the invention may beconjugated or otherwise associated with biocompatible modifiers that maybe used to adjust, alter, improve or moderate modulator characteristicsas desired. For example, antibodies or fusion constructs with increasedin vivo half-lives can be generated by attaching relatively highmolecular weight polymer molecules such as commercially availablepolyethylene glycol (PEG) or similar biocompatible polymers. Thoseskilled in the art will appreciate that PEG may be obtained in manydifferent molecular weight and molecular configurations that can beselected to impart specific properties to the antibody (e.g. thehalf-life may be tailored). PEG can be attached to modulators orantibody fragments or derivatives with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus of said antibodies or antibody fragments or via epsilon-aminogroups present on lysine residues. Linear or branched polymerderivatization that results in minimal loss of biological activity maybe used. The degree of conjugation can be closely monitored by SDS-PAGEand mass spectrometry to ensure optimal conjugation of PEG molecules toantibody molecules. Unreacted PEG can be separated from antibody-PEGconjugates by, e.g., size exclusion or ion-exchange chromatography. In asimilar manner, the disclosed modulators can be conjugated to albumin inorder to make the antibody or antibody fragment more stable in vivo orhave a longer half life in vivo. The techniques are well known in theart, see e.g., International Publication Nos. WO 93/15199, WO 93/15200,and WO 01/77137; and European Patent No. 0 413, 622. Other biocompatibleconjugates are evident to those of ordinary skill and may readily beidentified in accordance with the teachings herein.

D. Diagnostic or Detection Agents

In other preferred embodiments, modulators of the present invention, orfragments or derivatives thereof, are conjugated to a diagnostic ordetectable agent, marker or reporter which may be, for example, abiological molecule (e.g., a peptide or nucleotide), a small molecule,fluorophore, or radioisotope. Labeled modulators can be useful formonitoring the development or progression of a hyperproliferativedisorder or as part of a clinical testing procedure to determine theefficacy of a particular therapy including the disclosed modulators(i.e. theragnostics) or to determine a future course of treatment. Suchmarkers or reporters may also be useful in purifying the selectedmodulator, modulator analytics (e.g., epitope binding or antibodybinning), separating or isolating TIC or in preclinical procedures ortoxicology studies.

Such diagnosis analysis and/or detection can be accomplished by couplingthe modulator to detectable substances including, but not limited to,various enzymes comprising for example horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; prostheticgroups, such as but not limited to streptavidinibiotin andavidin/biotin; fluorescent materials, such as but not limited to,umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as but not limitedto iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium(³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In,), and technetium (⁹⁹Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and¹¹⁷Tin; positron emitting metals using various positron emissiontomographies, noradioactive paramagnetic metal ions, and molecules thatare radiolabeled or conjugated to specific radioisotopes. In suchembodiments appropriate detection methodology is well known in the artand readily available from numerous commercial sources.

As indicated above, in other embodiments the modulators or fragmentsthereof can be fused or conjugated to marker sequences or compounds,such as a peptide or fluorophore to facilitate purification ordiagnostic or analytic procedures such as immunohistochemistry,bio-layer interferometry, surface plasmon resonance, flow cytometry,competitive ELISA, FACs, etc. In preferred embodiments, the markercomprises a his-tag such as that provided by the pQE vector (Qiagen),among others, many of which are commercially available. Other peptidetags useful for purification include, but are not limited to, thehemagglutinin “HA” tag, which corresponds to an epitope derived from theinfluenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) andthe “flag” tag (U.S. Pat. No. 4,703,004).

E. Therapeutic Moieties

As previously alluded to the modulators or fragments or derivativesthereof may also be conjugated, linked or fused to or otherwiseassociated with a “therapeutic moiety” or “drug” such as ananti-proliferative or anti-cancer agent including, but not limited to,cytotoxic agents, cytostatic agents, anti-angiogenic agents, debulkingagents, chemotherapeutic agents, radiotherapy and radiotherapeuticagents, targeted anti-cancer agents, BRMs, therapeutic antibodies,cancer vaccines, cytokines, hormone therapies, radiation therapy andanti-metastatic agents and immunotherapeutic agents.

Preferred exemplary anti-cancer agents include cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxoruhicin,daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4(Immunogen, Inc.), dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, epirubicin, and cyclophosphamide and analogs orhomologs thereof. Additional compatible cytotoxins comprise dolastatinsand auristatins, including monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF) (Seattle Genetics, Inc.), amanitins such asalpha-amanitin, beta-amanitin, gamma-amanitin or epsilon-amanitin(Heidelberg Pharma AG), DNA minor groove binding agents such asduocarmycin derivatives (Syntarga, B.V.) and modifiedpyrrolobenzodiazepine dimers (Spirogen, Ltd.), splicing inhibitors suchas meayamycin analogs or derivatives (e.g., FR901464 as set forth inU.S. Pat. No. 7,825,267), tubular binding agents such as epothiloneanalogs and paclitaxel and DNA damaging agents such as calicheamicinsand esperamicins. Furthermore, in certain embodiments the DLL3modulators of the instant invention may be associated with anti-CD3binding molecules to recruit cytotoxic T-cells and have them target thetumor initiating cells (BiTE technology; see e.g., Fuhrmann, S. et. al.Annual Meeting of AACR Abstract No. 5625 (2010) which is incorporatedherein by reference).

Still additional compatible anti-cancer agents include, but are notlimited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa, chlorambucil, melphalan,carmustine (BCNU) and lomustine (CCNU), busulfan, dibromomannitol,streptozotocin, and cisdichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,vineristine and vinblastine). A more extensive list of therapeuticmoieties can be found in PCT publication WO 03/075957 and U.S.P.N.2009/0155255 each of which is incorporated herein by reference.

As indicated above selected embodiments of the instant invention aredirected to conjugated DLL3 modulators such as anti-DLL3 antibody drugconjugates that comprise pyrrolobenzodiazepine (PBD) as a cytotoxieagent. It will be appreciated that PBDs are alkylating agents that exertantitumor activity by covalently binding to DNA in the minor groove andinhibiting nucleic acid synthesis. In this respect PBDs have been shownto have potent antitumor properties while exhibiting minimal bone marrowdepression. PBDs compatible with the present invention may be linked tothe DLL3 modulator using any one of several types of linker (e.g., apeptidyl linker comprising a maleimido moiety with a free sulthydryl)and, in certain embodiments are dimeric in form (i.e., PBD dimers).Compatible PBDs (and optional linkers) that may be conjugated to thedisclosed modulators are described, for example, in U.S. Pat. Nos.6,362,331, 7,049,311, 7,189,710, 7,429,658, 7,407,951, 7,741,319,7,557,099, 8,034,808, 8,163,736 U.S.P.N, 2011/0256157 and PCT filingsWO2011/130613, WO2011/128650 and WO2011/130616 each of which isincorporated herein by reference. Accordingly, in particularly preferredembodiments the modulator will comprise an anti DLL3 antibody conjugatedor associated with one or more PBD dimers (i.e., a DLL3-PBD ADC).

In particularly preferred embodiments compatible PBDs that may beconjugated to the disclosed modulators are described, in U.S.P.N.2011/0256157. In this disclosure, PBD dimers, i.e. those comprising twoPBD moieties may be preferred. Thus, preferred conjugates of the presentinvention are those having the formulae (AB) or (AC):

wherein:

the dotted lines indicate the optional presence of a double bond betweenC1 and C2 or C2 and C3;

R² is independently selected from H, OH, ═O, ═CH₂, CN, R, OR, ═CH-R^(D),═C(R^(D))₂, O—SO₂-R, CO₂R and COR, and optionally further selected fromhalo or dihalo;

where R^(D) is independently selected from R, CO₂R, COR, CHO, CO₂H, andhalo;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, NO₂, Me₃Sn and halo;

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo;

R¹⁰ is a linker connected to a modulator or fragment or derivativethereof, as described above;

Q is independently selected from O, S and NH;

R¹¹ is either H, or R or, where Q is O, SO₃M, where M is a metal cation;

R and R′ are each independently selected from optionally substitutedC₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups, and optionally inrelation to the group NRR′, R and R′ together with the nitrogen atom towhich they are attached form an optionally substituted 4-, 5-, 6- or7-membered heterocyclic ring; and

wherein R^(2″), R^(6″), R^(7″), R^(9″), X″, Q″ and R^(11″) and are asdefined according to R², R⁶ R⁷, R⁹, X, Q and R¹¹ respectively, and R^(C)is a capping group.

Double Bond

In one embodiment, there is no double bond present between C1 and C2,and C2 and C3.

In one embodiment, the dotted lines indicate the optional presence of adouble bond between C2 and C3, as shown below:

In one embodiment, a double bond is present between C2 and C3 when R² isC₅₋₂₀ aryl or C₁₋₁₂ alkyl.

In one embodiment, the dotted lines indicate the optional presence of adouble bond between C1 and C2, as shown below:

In one embodiment, a double bond is present between C1 and C2 when R² isC₅₋₂₀ aryl or C₁₋₁₂ alkyl.

R²

In one embodiment, R² is independently selected from H, OH, ═O, ═CH₂,CN, R, OR, ═CH-R^(D), ═C(R^(D))₂, O—SO₂-R, CO₂R and COR, and optionallyfurther selected from halo or dihalo.

In one embodiment, R² is independently selected from H, OH, ═O, ═CH₂,CN, R, OR, ═CH-R^(D), ═C(R^(D))₂, O═SO₂-R, CO₂R and COR.

In one embodiment, R² is independently selected from H, ═O, ═CH₂, R,═CH-R^(D), and ═C(R^(D))₂.

In one embodiment, R² is independently H.

In one embodiment, R² is independently ═O.

In one embodiment, R² is independently —CH₂.

In one embodiment, R² is independently —CH-R^(D). Within the PBDcompound, the group ═CH-R^(D) may have either configuration shown below:

In one embodiment, the configuration is configuration (I).

In one embodiment, R² is independently ═C(R^(D))₂.

In one embodiment, R² is independently ═CF₂.

In one embodiment, R² is independently R.

In one embodiment, R² is independently optionally substituted C₅₋₂₀aryl.

In one embodiment, R² is independently optionally substituted C₁₋₁₂alkyl.

In one embodiment, R² is independently optionally substituted C₅₋₂₀aryl.

In one embodiment, R² is independently optionally substituted C₅₋₇ aryl.

In one embodiment, R² is independently optionally substituted C₈₋₁₀aryl.

In one embodiment, R² is independently optionally substituted phenyl.

In one embodiment, R² is independently optionally substituted napthyl.

In one embodiment, R² is independently optionally substituted pyridyl.

In one embodiment, R² is independently optionally substituted quinolinylor isoquinolinyl.

In one embodiment, R² bears one to three substituent groups, with 1 and2 being more preferred, and singly substituted groups being mostpreferred. The substituents may be any position.

Where R² is a C₅₋₇ aryl group, a single substituent is preferably on aring atom that is not adjacent the bond to the remainder of thecompound, i.e. it is preferably β or γ to the bond to the remainder ofthe compound. Therefore, where the C₅₋₇ aryl group is phenyl, thesubstituent is preferably in the meta- or para-positions, and morepreferably is in the para-position.

In one embodiment, R² is selected from:

where the asterisk indicates the point of attachment.

Where R² is a C₈₋₁₀ aryl group, for example quinolinyl or isoquinolinyl,it may bear any number of substituents at any position of the quinolineor isoquinoline rings. In some embodiments, it bears one, two or threesubstituents, and these may be on either the proximal and distal ringsor both (if more than one substituent).

In one embodiment, where R² is optionally substituted, the substituentsare selected from those substituents given in the substituent sectionbelow.

Where R is optionally substituted, the substituents are preferablyselected from:

Halo, Hydroxyl, Ether, Formyl, Acyl, Carboxy, Ester, Acyloxy, Amino,Amido, Acylamido, Aminocarbonyloxy, Ureido, Nitro, Cyano and Thioether.

In one embodiment, where R or R² is optionally substituted, thesubstituents are selected from the group consisting of R, OR, SR, NRR′,NO₂, halo, CO₂R, COR, CONH₂, CONHR, and CONRR′.

Where R² is C₁₋₁₂ alkyl, the optional substituent may additionallyinclude C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups.

Where R² is C₃₋₂₀ heterocyclyl, the optional substituent mayadditionally include C₁₋₁₂ alkyl and C₅₋₂₀ aryl groups.

Where R² is C₅₋₂₀ aryl groups, the optional substituent may additionallyinclude C₃₋₂₀ heterocyclyl and C₁₋₁₂ alkyl groups.

It is understood that the term “alkyl” encompasses the sub-classesalkenyl and alkynyl as well as cycloalkyl. Thus, where R² is optionallysubstituted C₁₋₁₂ alkyl, it is understood that the alkyl groupoptionally contains one or more carbon-carbon double or triple bonds,which may form part of a conjugated system. In one embodiment, theoptionally substituted C₁₋₁₂ alkyl group contains at least onecarbon-carbon double or triple bond, and this bond is conjugated with adouble bond present between C1 and C2, or C2 and C3. In one embodiment,the C₁₋₁₂ alkyl group is a group selected from saturated C₁₋₁₂ alkyl,C₂₋₁₂ alkenyl, C₂₋₁₂ alkynyl and C₃₋₁₂ cycloalkyl.

If a substituent on R² is halo, it is preferably F or Cl, morepreferably Cl.

If a substituent on R² is ether, it may in some embodiments be an alkoxygroup, for example, a C₁₋₇ alkoxy group (e.g. methoxy, ethoxy) or it mayin some embodiments be a C₅₋₇ aryloxy group (e.g phenoxy, pyridyloxy,furanyloxy).

If a substituent on R² is C₁₋₇ alkyl, it may preferably be a C₁₋₄ alkylgroup (e.g. methyl, ethyl, propyl, butyl).

If a substituent on R² is C₃₋₇ heterocyclyl, it may in some embodimentsbe C₆ nitrogen containing heterocyclyl group, e.g. morpholino,thiomorpholino, piperidinyl, piperazinyl. These groups may be bound tothe rest of the PBD moiety via the nitrogen atom. These groups may befurther substituted, for example, by C₁₋₄ alkyl groups.

If a substituent on R² is bis-oxy-C₁₋₃ alkylene, this is preferablybis-oxy-methylene or bis-oxy-ethylene.

Particularly preferred substituents for R² include methoxy, ethoxy,fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholinoand methyl-thienyl.

Particularly preferred substituted R² groups include, but are notlimited to, 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl,3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl,3,4-bisoxymethylene-phenyl, 4-methylthienyl, 4-cyanophenyl,4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl andisoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, and naphthyl.

In one embodiment, R² is halo or dihalo. In one embodiment, R² is —F or—F₂, which substituents are illustrated below as (III) and (IV)respectively:

R^(D)

In one embodiment, R^(D) is independently selected from R, CO₂R, COR,CHO, CO₂H, and halo.

In one embodiment, R^(D) is independently R.

In one embodiment, R^(D) is independently halo.

R⁶

In one embodiment, R⁶ is independently selected from H, R, OH, OR, SH,SR, NH₂, NHR, NRR′, NO₂, Me₃Sn— and Halo.

In one embodiment, R⁶ is independently selected from H, OH, OR, SH, NH₂,NO₂ and Halo.

In one embodiment, R⁶ is independently selected from H and Halo.

In one embodiment, R⁶ is independently H.

In one embodiment, R⁶ and R⁷ together form a group —O—(CH₂)_(p)—O—,where p is 1 or 2.

R⁷

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′,NO₂, Me₃Sn and halo.

In one embodiment, R⁷ is independently OR.

In one embodiment, R⁷ is independently OR^(7A), where R^(7A) isindependently optionally substituted C₁₋₆ alkyl.

In one embodiment, R^(7A) is independently optionally substitutedsaturated C₁₋₆ alkyl.

In one embodiment, R^(7A) is independently optionally substituted C₂₋₄alkenyl.

In one embodiment, R^(7A) is independently Me.

In one embodiment, R^(7A) is independently CH₂Ph.

In one embodiment, R^(7A) is independently allyl.

In one embodiment, the compound is a dimer where the R⁷ groups of eachmonomer form together a dimer bridge having the formula X-R″-X linkingthe monomers.

R⁸

In one embodiment, the compound is a dimer where the R⁸ groups of eachmonomer form together a dimer bridge having the formula X-R″-X linkingthe monomers.

In one embodiment, R⁸ is independently OR^(8A), where R^(8A) isindependently optionally substituted C₁₋₄ alkyl.

In one embodiment, R^(8A) is independently optionally substitutedsaturated C₁₋₆ alkyl or optionally substituted C₂₋₄ alkenyl.

In one embodiment, R^(8A) is independently Me.

In one embodiment, R^(8A) is independently CH₂Ph.

In one embodiment, R^(8A) is independently allyl.

In one embodiment, R⁸ and R⁷ together form a group —O—(CH₂)_(p)—O—,where p is 1 or 2.

In one embodiment, R⁸ and R⁹ together form a group —O—(CH₂)_(p)—O—,where p is 1 or 2.

R⁹

In one embodiment, R⁹ is independently selected from H, R, OH, OR, SH,SR, NH₂, NHR, NRR′, NO₂, Me₃Sn— and Halo.

In one embodiment, R⁹ is independently H.

In one embodiment, R⁹ is independently R or OR.

R and R′

In one embodiment, R is independently selected from optionallysubstituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups. Thesegroups are each defined in the substituents section below.

In one embodiment, R is independently optionally substituted C₁₋₁₂alkyl.

In one embodiment, R is independently optionally substituted C₃₋₂₀heterocyclyl.

In one embodiment, R is independently optionally substituted C₅₋₂₀ aryl.

In one embodiment, R is independently optionally substituted C₁₋₁₂alkyl.

Described above in relation to R² are various embodiments relating topreferred alkyl and aryl groups and the identity and number of optionalsubstituents. The preferences set out for R² as it applies to R areapplicable, where appropriate, to all other groups R, for examples whereR⁶, R⁷, R⁸ or R⁹ is R.

The preferences for R apply also to R′.

In some embodiments of the invention there is provided a compound havinga substituent group —NRR′. In one embodiment, R and R′ together with thenitrogen atom to which they are attached form an optionally substituted4-, 5-, 6- or 7-membered heterocyclic ring. The ring may contain afurther heteroatom, for example N, O or S.

In one embodiment, the heterocyclic ring is itself substituted with agroup R. Where a further N heteroatom is present, the substituent may beon the N heteroatom.

R″

R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g.benzene or pyridine, which rings are optionally substituted.

In one embodiment, R″ is a C₃₋₁₂ alkylene group, which chain may beinterrupted by one or more heteroatoms and/or aromatic rings, e.g.benzene or pyridine.

In one embodiment, the alkylene group is optionally interrupted by oneor more heteroatoms selected from O, S, and NMe and/or aromatic rings,which rings are optionally substituted.

In one embodiment, the aromatic ring is a C₅₋₂₀ arylene group, wherearylene pertains to a divalent moiety obtained by removing two hydrogenatoms from two aromatic ring atoms of an aromatic compound, which moietyhas from 5 to 20 ring atoms.

In one embodiment, R″ is a C₃₋₁₂ alkylene group, which chain may beinterrupted by one or more heteroatoms, e.g. O, S, N(H), NMe and/oraromatic rings, e.g. benzene or pyridine, which rings are optionallysubstituted by NH₂.

In one embodiment, R″ is a C₃₋₁₂ alkylene group.

In one embodiment, R″ is selected from a C₃, C₅, C₇, C₉ and a C₁₁alkylene group.

In one embodiment, R″ is selected from a C₃, C₅ and a C₇ alkylene group.

In one embodiment, R″ is selected from a C₃ and a C₅ alkylene group.

In one embodiment, R″ is a C₃ alkylene group.

In one embodiment, R″ is a C₅ alkylene group.

The alkylene groups listed above may be optionally interrupted by one ormore heteroatoms and/or aromatic rings, e.g. benzene or pyridine, whichrings are optionally substituted.

The alkylene groups listed above may be optionally interrupted by one ormore heteroatoms and/or aromatic rings, e.g. benzene or pyridine.

The alkylene groups listed above may be unsubstituted linear aliphaticalkylene groups.

X

In one embodiment, X is selected from O, S, N(H).

Preferably, X is O.

R¹⁰

Preferably compatible linkers such as those described above attach aDLL3 modulator (CBA/Ab/M), to a PBD drug moiety D through covalentbond(s) at the R¹⁰ position (i.e., N10). The linker is a bifunctional ormultifunctional moiety which can be used to link one or more drug moiety(D) and a modulator (preferably an antibody) to form antibody-drugconjugates (ADC). The linker (L) may be stable outside a cell, i.e.extracellular, or it may be cleavable by enzymatic activity, hydrolysis,or other metabolic conditions. Antibody-drug conjugates (ADC) can beconveniently prepared using a linker having reactive functionality forbinding to the drug moiety and to the antibody. A cysteine thiol, or anamine, e.g. N-terminus or amino acid side chain such as lysine, of theantibody (Ab) can form a bond with a functional group of a linker orspacer reagent, PBD drug moiety (D) or drug-linker reagent (D-L).

Many functional groups on the linker attached to the N10 position of thePBD moiety may be useful to react with the cell binding agent. Forexample, ester, thioester, amide, thioamide, carbamate, thiocarbamate,urea, thiourea, ether, thioether, or disulfide linkages may be formedfrom reaction of the linker-PBD drug intermediates and the cell bindingagent.

In another embodiment, the linker may be substituted with groups thatmodulate aggregation, solubility or reactivity. For example, a sulfonatesubstituent may increase water solubility of the reagent and facilitatethe coupling reaction of the linker reagent with the antibody or thedrug moiety, or facilitate the coupling reaction of Ab-L with D, or D-Lwith Ab, depending on the synthetic route employed to prepare the ADC.

In one preferred embodiment, R¹⁰ is a group:

where the asterisk indicates the point of attachment to the N10position, CBA is a cell binding agent/modulator, L¹ is a linker, A is aconnecting group connecting L¹ to the cell binding agent, L² is acovalent bond or together with —OC(═O)— forms a self-immolative linker,and L¹ or L² is a cleavable linker.

L¹ is preferably the cleavable linker, and may be referred to as atrigger for activation of the linker for cleavage.

As discussed in the linker section above the nature of L¹ and L², wherepresent, can vary widely. These groups are chosen on the basis of theircleavage characteristics, which may be dictated by the conditions at thesite to which the conjugate is delivered. Those linkers that are cleavedby the action of enzymes are preferred, although linkers that arecleavable by changes in pH (e.g. acid or base labile), temperature orupon irradiation (e.g. photolabile) may also be used. Linkers that arecleavable under reducing or oxidizing conditions may also find use inthe present invention.

L¹ may comprise a contiguous sequence of amino acids. The amino acidsequence may be the target substrate for enzymatic cleavage, therebyallowing release of R¹⁰ from the N10 position.

In one embodiment, L¹ is cleavable by the action of an enzyme. In oneembodiment, the enzyme is an esterase or a peptidase.

In one embodiment, L² is present and together with —C(═O)O— forms aself-immolative linker. In one embodiment, L² is a substrate forenzymatic activity, thereby allowing release of R¹⁰ from the N10position.

In one embodiment, where L¹ is cleavable by the action of an enzyme andL² is present, the enzyme cleaves the bond between L¹ and L².

With regard to attaching the chosen linker to a selected PBD the groupR^(C) is removable from the N10 position of certain PBD moieties toleave an N10-C11 imine bond, a carbinolamine, a substitutedcarbinolamine, where QR¹¹ is OSO₃M, a bisulfite adduct, athiocarbinolamine, a substituted thiocarbinoiamine, or a substitutedcarbinalamine.

In one embodiment, R^(C), may be a protecting group that is removable toleave an N10-C11 imine bond, a carbinolamine, a substitutedcabinolamine, or, where QR¹¹ is OSO₃M, a bisulfite adduct. In oneembodiment, R^(C) is a protecting group that is removable to leave anN10-C11 imine bond.

The group R^(C) is intended to be removable under the same conditions asthose required for the removal of the group R¹⁰, for example to yield anN10-C11 imine bond, a carbinolamine and so on. The capping group acts asa protecting group for the intended functionality at the N10 position.The capping group is intended not to be reactive towards a cell bindingagent. For example, R^(C) is not the same as R^(L).

Compounds having a capping group may be used as intermediates in thesynthesis of dimers having an imine monomer. Alternatively, compoundshaving a capping group may be used as conjugates, where the cappinggroup is removed at the target location to yield an imine, acarbinolamine, a substituted cabinolamine and so on. Thus, in thisembodiment, the capping group may be referred to as a therapeuticallyremovable nitrogen protecting group, as defined in WO 00/12507.

In one embodiment, the group R^(C) is removable under the conditionsthat cleave the linker R^(L) of the group R¹⁰. Thus, in one embodiment,the capping group is cleavable by the action of an enzyme.

In an alternative embodiment, the capping group is removable prior tothe connection of the linker R^(L) to the modulator. In this embodiment,the capping group is removable under conditions that do not cleave thelinker R^(L).

Where a compound includes a functional group G¹ to form a connection tothe cell binding agent, the capping group is removable prior to theaddition or unmasking of G¹.

The capping group may be used as part of a protecting group strategy toensure that only one of the monomer units in a dimer is connected to acell binding agent.

The capping group may be used as a mask for a N10-C11 imine bond. Thecapping group may be removed at such time as the imine functionality isrequired in the compound. The capping group is also a mask for acarbinolamine, a substituted cabinolamine, and a bisulfite adduct, asdescribed above.

In one embodiment, R^(C) is a carbamate protecting group.

In one embodiment, the carbamate protecting group is selected from:

Alloc, Fmoc, Boc, Troc, Teoc, Psec, Cbz and PNZ.

Optionally, the carbamate protecting group is further selected from Moc.

In one embodiment, R^(C) is a linker group R^(L) lacking the functionalgroup for connection to the cell binding agent.

This application is particularly concerned with those R^(C) groups whichare carbamates.

In one embodiment, R^(C) is a group:

where the asterisk indicates the point of attachment to the N10position, G² is a terminating group, L³ is a covalent bond or acleavable linker L¹, L² is a covalent bond or together with OC(═O) formsa self-immolative linker.

Where L³ and L² are both covalent bonds, G² and OC(═O) together form acarbamate protecting group as defined above.

L¹ is as defined above in relation to R¹⁰.

L² is as defined above in relation to R¹⁰.

Various terminating groups are described below, including those based onwell known protecting groups.

In one embodiment L³ is a cleavable linker L¹, and L², together withOC(═O), forms a self-immolative linker. In this embodiment, G² is Ac(acetyl) or Moc, or a carbamate protecting group selected from: Alloc,Fmoc, Boc, Troc, Teoc, Psec, Cbz and PNZ. Optionally, the carbamateprotecting group is further selected from Moc.

In another embodiment, G² is an acyl group —C(═O)G³, where G³ isselected from alkyl (including cycloalkyl, alkenyl and alkynyl),heteroalkyl, heterocyclyl and aryl (including heteroaryl and carboaryl).These groups may be optionally substituted. The acyl group together withan amino group of L³ or L², where appropriate, may form an amide bond.The acyl group together with a hydroxy group of L³ or L², whereappropriate, may form an ester bond.

In one embodiment, G³ is heteroalkyl. The heteroalkyl group may comprisepolyethylene glycol. The heteroalkyl group may have a heteroatom, suchas O or N, adjacent to the acyl group, thereby forming a carbamate orcarbonate group, where appropriate, with a heteroatom present in thegroup L³ or L², where appropriate.

In one embodiment, G³ is selected from NH₂, NHR and NRR′. Preferably, G³is NRR′.

In one embodiment G² is the group:

where the asterisk indicates the point of attachment to L³, n is 0 to 6and G⁴ is selected from OH, OR, SH, SR, COOR, CONH₂, CONHR, CONRR′, NH₂,NHR, NRR′, NO₂, and halo. The groups OH, SH, NH₂ and NHR are protected.In one embodiment, n is 1 to 6, and preferably n is 5. In oneembodiment, G⁴ is OR, SR, COOR, CONH₂, CONHR, CONRR′, and NRR′. In oneembodiment, G⁴ is OR, SR, and NRR′. Preferably G⁴ is selected from ORand NRR′, most preferably G⁴ is OR. Most preferably G⁴ is OMe.

In one embodiment, the group G² is:

where the asterisk indicates the point of attachment to L³, and n and G⁴are as defined above.

In one embodiment, the group G² is:

where the asterisk indicates the point of attachment to L³, n is 0 or 1,m is 0 to 50, and G⁴ is selected from OH, OR, SH, SR, COOR, CONH₂,CONHR, CONRR′, NH₂, NHR, NRR′, NO₂, and halo. In a preferred embodiment,n is 1 and m is 0 to 10, 1 to 2, preferably 4 to 8, and most preferably4 or 8. In another embodiment, n is 1 and m is 10 to 50, preferably 20to 40. The groups OH, SH, NH₂ and NHR are protected. In one embodiment,G⁴ is OR, SR, COOR, CONH₂, CONHR, CONRR′, and NRR′. In one embodiment,G⁴ is OR, SR, and NRR′. Preferably G⁴ is selected from OR and NRR′, mostpreferably G⁴ is OR. Preferably G⁴ is OMe.

In one embodiment, the group G² is:

where the asterisk indicates the point of attachment to L³, and n, m andG⁴ are as defined above.

In one embodiment, the group G² is:

where n is 1-20, m is 0-6, and G⁴ is selected from OH, OR, SH, SR, COOR,CONH₂, CONHR, CONRR′, NH₂, NHR, NRR′, NO₂, and halo. In one embodiment,n is 1-10. In another embodiment, n is 10 to 50, preferably 20 to 40. Inone embodiment, n is 1. In one embodiment, m is 1. The groups OH, SH,NH₂ and NHR are protected. In one embodiment, G⁴ is OR, SR, COOR, CONH₂,CONHR, CONRR′, and NRR′. In one embodiment, G⁴ is OR, SR, and NRR′.Preferably G⁴ is selected from OR and NRR′, most preferably G⁴ is OR.Preferably G⁴ is OMe.

In one embodiment, the group G² is:

where the asterisk indicates the point of attachment to L³, and n, m andG⁴ are as defined above.

In each of the embodiments above G⁴ may be OH, SH, NH₂ and NHR. Thesegroups are preferably protected.

In one embodiment, OH is protected with Bzl, TBDMS, or TBDPS.

In one embodiment, SH is protected with Acm, Bzl, Bzl-OMe, Bzl-OMe, orTrt.

In one embodiment, NH₂ or NHR are protected with Boc, Moc, Z-Cl, Fmoc,Z, or Alloc.

In one embodiment, the group G² is present in combination with a groupL³, which group is a dipeptide.

The capping group is not intended for connection to the modulator. Thus,the other monomer present in the dimer serves as the point of connectionto the modulator via a linker. Accordingly, it is preferred that thefunctionality present in the capping group is not available for reactionwith a modulator. Thus, reactive functional groups such as OH, SH, NH₂,COOH are preferably avoided. However, such functionality may be presentin the capping group if protected, as described above.

Thus, in accordance with the teachings herein one embodiment of theinvention comprises a conjugate comprising a compound:

wherein CBA is a cell binding agent/modulator, and n is 0 or 1. L¹ is aspreviously defined, and R^(E) and R^(E)″ are each independently selectedfrom H or R^(D).

In another embodiment, the conjugate comprises a compound:

wherein CBA is a cell binding agent/modulator, L¹ is as previouslydefined, Ar¹ and Ar² are each independently optionally substituted C₅₋₂₀aryl, and n is 0 or 1.

Those of skill in the art will appreciate that other symmetric andasymmetric PBD dimers and linkers are compatible with the instantinvention and could be selected without undue experimentation based onthe teachings herein and the prior art.

Another aspect of the invention includes ADCs comprising radioisotopes.Exemplary radioisotopes that may be compatible with such embodimentsinclude, but are not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I,),carbon (¹⁴C), copper (⁶²Cu, ⁶⁴Cu, ⁶⁷Cu), sulfur (³⁵S), tritium (³H),indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In,), bismuth (²¹²Bi, ²¹³Bi), technetium(⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd),molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd,¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶HO, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru,⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn,¹¹⁷Sn, ²²⁵Ac, ⁷⁶Br, and ²¹¹At. Other radionuclides are also available asdiagnostic and therapeutic agents, especially those in the energy rangeof 60 to 4,000 keV. Depending on the condition to be treated and thedesired therapeutic profile, those skilled in the art may readily selectthe appropriate radioisotope for use with the disclosed modulators.

DLL3 modulators of the present invention may also be conjugated to atherapeutic moiety or drug that modifies a given biological response(e.g., biological response modifiers or BRMs). That is, therapeuticagents or moieties compatible with the instant invention are not to beconstrued as limited to classical chemical therapeutic agents. Forexample, in particularly preferred embodiments the drug moiety may be aprotein or polypeptide or fragment thereof possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, Onconase (or another cytotoxic RNase),pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein suchas tumor necrosis factor, α-interferon, β-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator, anapoptotie agent, e.g., TNF-α, TNF-β, AIM I (see, InternationalPublication No. WO 97/33899), AIM II (see, International Publication No.WO 97/34911), Fas Ligand (Takahashi et al., 1994, J. Immunol., 6:1567),and VEGI (see, International Publication No. WO 99/23105), a thromboticagent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or,a biological response modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), or a growth factor(e.g., growth hormone (“GH”)). As set forth above, methods for fusing orconjugating modulators to polypeptide moieties are known in the art. Inaddition to the previously disclosed subject references see, e.g., U.S.Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851, and5,112,946; EP 307,434; EP 367,166; PCT Publications WO 96/04388 and WO91/06570; Ashkenazi et al., 1991, PNAS USA 88:10535; Zheng et al., 1995,J Immunol 154:5590; and Vil et al., 1992, PNAS USA 89:11337 each ofwhich is incorporated herein by reference. Moreover, as set forth abovethe association of a modulator with such moieties does not necessarilyneed to be direct, but may occur through linker sequences. As previouslyalluded to, such linker molecules are commonly known in the art anddescribed in Denardo et al., 1998, Clin Cancer Res 4:2483; Peterson etal., 1999, Bioconjug Chem 10:553; Zimmerman et al., 1999, Nucl Med Biol26:943; Garnett, 2002, Adv Drug Deliv Rev 53:171 each of which isincorporated herein.

IX. Diagnostics and Screening

A. Diagnostics

In yet other embodiments, the invention provides in vitro or in vivomethods for detecting, diagnosing or monitoring proliferative disordersand methods of screening cells from a patient to identify tumorigeniccells including CSCs. Such methods include identifying an individualhaving cancer for treatment or monitoring progression of a cancercomprising contacting the patient or a sample obtained from a patient(i.e. either in vivo or in vitro) with a modulator as described hereinand detecting presence or absence, or level of association, of themodulator to bound or free target molecules in the sample. Inparticularly preferred embodiments the modulator will comprise adetectable label or reporter molecule as described herein.

In some embodiments, the association of the modulator, such as anantibody, with particular cells in the sample likely denotes that thesample may contain CSCs, thereby indicating that the individual havingcancer may be effectively treated with a modulator as described herein.The methods may further comprise a step of comparing the level ofbinding to a control. Conversely, when the modulator is a Fc-construct,the binding properties may be exploited and monitored (directly orindirectly, in vivo or in vitro) when in contact with the sample toprovide the desired information.

Exemplary compatible assay methods include radioimmunoassays, enzymeimmunoassays, competitive-binding assays, fluorescent immunoassay,immunoblot assays, Western Blot analysis, flow cytometry assays, andELISA assays. Compatible in vivo theragnostics or diagnostics maycomprise art-recognized imaging or monitoring techniques such asmagnetic resonance imaging, computerized tomography (e.g. CAT scan),positron tomography (e.g., PET scan) radiography, ultrasound, etc., aswould be known by those skilled in the art.

In another embodiment, the invention provides a method of analyzingcancer progression and/or pathogenesis in vivo. In another embodiment,analysis of cancer progression and/or pathogenesis in vivo comprisesdetermining the extent of tumor progression. In another embodiment,analysis comprises the identification of the tumor. In anotherembodiment, analysis of tumor progression is performed on the primarytumor. In another embodiment, analysis is performed over time dependingon the type of cancer as known to one skilled in the art. In anotherembodiment, further analysis of secondary tumors originating frommetastasizing cells of the primary tumor is analyzed in-vivo. In anotherembodiment, the size and shape of secondary tumors are analyzed. In someembodiments, further ex vivo analysis is performed.

In another embodiment, the invention provides a method of analyzingcancer progression and/or pathogenesis in vivo including determiningcell metastasis or detecting and quantifying the level of circulatingtumor cells. In yet another embodiment, analysis of cell metastasiscomprises determination of progressive growth of cells at a site that isdiscontinuous from the primary tumor. In another embodiment, the site ofcell metastasis analysis comprises the route of neoplastic spread. Insome embodiment, cells can disperse via blood vasculature, lymphatics,within body cavities or combinations thereof. In another embodiment,cell metastasis analysis is performed in view of cell migration,dissemination, extravasation, proliferation or combinations thereof.

Accordingly, in a particularly preferred embodiment the modulators ofthe instant invention may be used to detect and quantify DLL3 levels ina patient sample (e.g., plasma or blood) which may, in turn, be used todetect, diagnose or monitor DLL3 associated disorders includingproliferative disorders. In related embodiments the modulators of theinstant invention may be used to detect, monitor and/or quantifycirculating tumor cells either in vivo or in vitro (see, for example, WO2012/0128801 which is incorporated herein by reference). In still otherpreferred embodiments the circulating tumor cells may comprise cancerstem cells.

In certain examples, the tumorigenic cells in a subject or a sample froma subject may be assessed or characterized using the disclosedmodulators prior to therapy or regimen to establish a baseline. In otherexamples the sample is derived from a subject that was treated. In someexamples the sample is taken from the subject at least about 1, 2, 4, 6,7, 8, 10, 12, 14, 15, 16, 18, 20, 30, 60, 90 days, 6 months, 9 months,12 months, or >12 months after the subject begins or terminatestreatment in certain examples, the tumorigenic cells are assessed orcharacterized after a certain number of doses (e.g., after 2, 5, 10, 20,30 or more doses of a therapy). In other examples, the tumorigenic cellsare characterized or assessed after 1 week, 2 weeks, 1 month, 2 months,1 year, 2 years, 3 years, 4 years or more after receiving one or moretherapies.

In another aspect, and as discussed in more detail below, the presentinvention provides kits for detecting, monitoring or diagnosing ahyperproliferative disorder, identifying individual having such adisorder for possible treatment or monitoring progression (orregression) of the disorder in a patient, wherein the kit comprises amodulator as described herein, and reagents for detecting the impact ofthe modulator on a sample.

Yet another aspect of the instant invention comprises the use of labeledDLL3 for immunohistochemistry (IHC). In this respect DLL3 IHC may beused as a diagnostic tool to aid in the diagnosis of variousproliferative disorders and to monitor the potential response totreatments including DLL3 modulator therapy. Compatible diagnosticassays may be performed on tissues that have been chemically fixed(including but not limited to: formaldehyde, gluteraldehyde, osmiumtetroxide, potassium dichromate, acetic acid, alcohols, zinc salts,mercuric chloride, chromium tetroxide and picric acid) and embedded(including but not limited to: glycol methacrylate, paraffin and resins)or preserved via freezing. As discussed in more detail below such assayscould be used to guide treatment decisions and determine dosing regimensand timing.

B. Screening

In certain embodiments, the modulators can also be used to screen for oridentify compounds or agents (e.g., drugs) that alter a function oractivity of tumorigenic cells or progeny thereof by interacting with anantigen (e.g., genotypic or phenotypic components thereof). Suchcompounds and agents can be drug candidates that are screened for thetreatment of a proliferative disorder, for example. In one embodiment, asystem or method includes tumorigenic cells comprising DLL3 and acompound or agent (e.g., drug), wherein the cells and compound or agentare in contact with each other. In such embodiments the subject cellsmay have been identified, monitored and/or enriched using the disclosedmodulators.

In yet another embodiment, a method includes contacting, directly orindirectly, tumorigenic cells or progeny thereof with a test agent orcompound and determining if the test agent or compound modulates anactivity or function of the antigen-associated tumorigenic cells. Oneexample of a direct interaction is physical interaction, while anindirect interaction includes the action of a composition upon anintermediary molecule that, in turn, acts upon the referenced entity(e.g., cell or cell culture). Exemplary activities or functions that canbe modulated include changes in cell morphology or viability, expressionof a marker, differentiation or de-differentiation, cell respiration,mitochondrial activity, membrane integrity, maturation, proliferation,viability, apoptosis or cell death.

Methods of screening and identifying agents and compounds include thosesuitable for high throughput screening, which include arrays of cells(e.g., microarrays) positioned or placed, optionally at pre-determinedlocations or addresses. For example, cells can be positioned or placed(pre-seeded) on a culture dish, tube, flask, roller bottle or plate(e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96,384 and 1536 multi-well plate or dish). High-throughput robotic ormanual handling methods can probe chemical interactions and determinelevels of expression of many genes in a short period of time. Techniqueshave been developed that utilize molecular signals (e.g., viafluorophores) and automated analyses that process information at a veryrapid rate (see, e.g., Pinhasov et al., Comb. Chem. High ThroughputScreen. 7:133 (2004)). For example, microarray technology has beenextensively used to probe the interactions of thousands of genes atonce, while providing information for specific genes (see, e.g.,Mocellin and Rossi, Adv. Exp. Med. Biol. 593:19 (2007)).

Libraries that can be screened include, for example, small moleculelibraries, phage display libraries, fully human antibody yeast displaylibraries (Adimab, LLC), siRNA libraries, and adenoviral transfectionvectors.

X. Pharmaceutical Preparations and Therapeutic Uses

A. Formulations and Routes of Administration

Depending on the form of the modulator along with any optionalconjugate, the mode of intended delivery, the disease being treated ormonitored and numerous other variables, compositions of the inventionmay be formulated as desired using art-recognized techniques. In someembodiments, the therapeutic compositions of the invention may beadministered neat or with a minimum of additional components whileothers may optionally be formulated to contain suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries that are wellknown in the art (see, e.g., Gennaro, Remington: The Science andPractice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20thed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7^(th) ed., Lippencott Williams and Wilkins (2004); Kibbe etal., Handbook of Pharmaceutical Excipients, 3^(rd) ed., PharmaceuticalPress (2000)). Various pharmaceutically acceptable carriers, whichinclude vehicles, adjuvants, and diluents, are readily available fromnumerous commercial sources. Moreover, an assortment of pharmaceuticallyacceptable auxiliary substances, such as pH adjusting and bufferingagents, tonicity adjusting agents, stabilizers, wetting agents and thelike, are also available. Certain non-limiting exemplary carriersinclude saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof.

More particularly it will be appreciated that, in some embodiments, thetherapeutic compositions of the invention may be administered neat orwith a minimum of additional components. Conversely the DLL3 modulatorsof the present invention may optionally be formulated to containsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries that are well known in the art and are relatively inertsubstances that facilitate administration of the modulator or which aidprocessing of the active compounds into preparations that arepharmaceutically optimized for delivery to the site of action. Forexample, an excipient can give form or consistency or act as a diluentto improve the pharmacokinetics or stability of the modulator. Suitableexcipients or additives include, but are not limited to, stabilizingagents, wetting and emulsifying agents, salts for varying osmolarity,encapsulating agents, buffers, and skin penetration enhancers. Incertain preferred embodiments the pharmaceutical compositions may beprovided in a lyophilized form and reconstituted in, for example,buffered saline prior to administration.

Disclosed modulators for systemic administration may be formulated forenteral, parenteral or topical administration. Indeed, all three typesof formulation may be used simultaneously to achieve systemicadministration of the active ingredient. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000). Suitable formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form,for example, water-soluble salts. In addition, suspensions of the activecompounds as appropriate for oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, hexylsubstituted poly(lactide), sesame oil, orsynthetic fatty acid esters, for example, ethyl oleate or triglycerides.Aqueous injection suspensions may contain substances that increase theviscosity of the suspension and include, for example, sodiumcarboxymethyl cellulose, sorbitol, and/or dextran. Optionally, thesuspension may also contain stabilizers. Liposomes can also be used toencapsulate the agent for delivery into the cell.

Suitable formulations for enteral administration include hard or softgelatin capsules, pills, tablets, including coated tablets, elixirs,suspensions, syrups or inhalations and controlled release forms thereof.

In general the compounds and compositions of the invention, comprisingDLL3 modulators may be administered in vivo, to a subject in needthereof, by various routes, including, but not limited to, oral,intravenous, intra-arterial, subcutaneous, parenteral, intranasal,intramuscular, intracranial, intracardiac, intraventricular,intratracheal, buccal, rectal, intraperitoneal, intradermal, topical,transdermal, and intrathecal, or otherwise by implantation orinhalation. The subject compositions may be formulated into preparationsin solid, semi-solid, liquid, or gaseous firms; including, but notlimited to, tablets, capsules, powders, granules, ointments, solutions,suppositories, enemas, injections, inhalants, and aerosols. Theappropriate formulation and route of administration may be selectedaccording to the intended application and therapeutic regimen.

B. Dosages

Similarly, the particular dosage regimen, i.e., dose, timing andrepetition, will depend on the particular individual and thatindividual's medical history, as well as empirical considerations suchas pharmacokinetics (e.g., half-life, clearance rate, etc.). Frequencyof administration may be determined and adjusted over the course oftherapy, and is based on reducing the number of proliferative ortumorigenic cells, maintaining the reduction of such neoplastic cells,reducing the proliferation of neoplastic cells, or delaying thedevelopment of metastasis. In other embodiments the dosage administeredmay be adjusted or attenuated to manage potential side effects and/ortoxicity. Alternatively, sustained continuous release formulations of asubject therapeutic composition may be appropriate.

In general, the modulators of the invention may be administered invarious ranges. These include about 10 μg/kg body weight to about 100mg/kg body weight per dose; about 50 μg/kg body weight to about 5 mg/kgbody weight per dose; about 100 μg/kg body weight to about 10 mg/kg bodyweight per dose. Other ranges include about 100 μg/kg body weight toabout 20 mg/kg body weight per dose and about 0.5 mg/kg body weight toabout 20 mg/kg body weight per dose. In certain embodiments, the dosageis at least about 100 μg/kg body weight, at least about 250 μg/kg bodyweight, at least about 750 μg/kg body weight, at least about 3 mg/kgbody weight, at least about 5 mg/kg body weight, at least about 10 mg/kgbody weight.

In selected embodiments the modulators will be administered atapproximately 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 μg/kg bodyweight per dose. Other embodiments will comprise the administration ofmodulators at 200, 300, 400, 500, 600, 700, 800 or 900 μg/kg body weightper dose. In other preferred embodiments the disclosed modulators willbe administered at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg. In still otherembodiments the modulators may be administered at 12, 14, 16, 18 or 20mg/kg body weight per dose. In yet other embodiments the modulators maybe administered at 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or100 mg/kg body weight per dose. In accordance with the teachings hereinit will be appreciated that the aforementioned dosages are applicable toboth unconjugated modulators and modulators conjugated to a cytotoxicagent. One of skill in the art could readily determine appropriatedosages for various conjugated and unconjugated modulators based onpreclinical animal studies, clinical observations and standard medicaland biochemical techniques and measurements.

With regard to conjugated modulators particularly preferred embodimentswill comprise dosages of between about 50 μg/kg to about 5 mg/kg bodyweight per dose. In this regard conjugated modulators may beadministered at 50, 75 or 100 μg/kg or at 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9 or 1 mg/kg body weight per dose. In other preferred embodimentsthe conjugated modulators of the instant invention may be administeredat 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25,4.5, 4.75 or 5 mg/kg body weight per dose. In particularly preferredembodiments such conjugated modulator dosages will be administeredintravenously over a period of time. Moreover, such dosages may beadministered multiple times over a defined course of treatment.

Other dosing regimens may be predicated on Body Surface Area (BSA)calculations as disclosed in U.S. Pat. No. 7,744,877. As is well known,the BSA is calculated using the patient's height and weight and providesa measure of a subject's size as represented by the surface area of hisor her body. In certain embodiments, the modulators may be administeredin dosages from 10 mg/m² to 800 mg/m², from 50 mg/m² to 500 mg/m² and atdosages of 100 mg/m², 150 mg/m², 200 mg/m², 250 mg/m², 300 mg/m², 350mg/m², 400 mg/m² or 450 mg/m².

It will also be appreciated that art recognized and empirical techniquesmay be used to determine appropriate dosage for conjugated modulators(i.e., ADCs).

In any event, DLL3 modulators (both conjugated and unconjugated) arepreferably administered as needed to subjects in need thereof.Determination of the frequency of administration may be made by personsskilled in the art, such as an attending physician based onconsiderations of the condition being treated, age of the subject beingtreated, severity of the condition being treated, general state ofhealth of the subject being treated and the like. Generally, aneffective dose of the DLL3 modulator is administered to a subject one ormore times. More particularly, an effective dose of the modulator isadministered to the subject once a month, more than once a month, orless than once a month. In certain embodiments, the effective dose ofthe DLL3 modulator may be administered multiple times, including forperiods of at least a month, at least six months, at least a year, atleast two years or a period of several years. In yet other embodiments,several days (2, 3, 4, 5, 6 or 7), several weeks (1, 2, 3, 4, 5, 6, 7 or8) or several months (1, 2, 3, 4, 5, 6, 7 or 8) or even a year orseveral years may lapse between administration of the disclosedmodulators.

In certain preferred embodiments the course of treatment involvingconjugated modulators will comprise multiple doses of the selected drugproduct (i.e., an ADC) over a period of weeks or months. Morespecifically, conjugated modulators of the instant invention mayadministered once every day, every two days, every four days, everyweek, every ten days, every two weeks, every three weeks, every month,every six weeks, every two months, every ten weeks or every threemonths. In this regard it will be appreciated that the dosages may bealtered or the interval may be adjusted based on patient response andclinical practices.

Dosages and regimens may also be determined empirically for thedisclosed therapeutic compositions in individuals who have been givenone or more administration(s). For example, individuals may be givenincremental dosages of a therapeutic composition produced as describedherein. In selected embodiments the dosage may be gradually increased orreduced or attenuated based respectively on empirically determined orobserved side effects or toxicity. To assess efficacy of the selectedcomposition, a marker of the specific disease, disorder or condition canbe followed as described previously. In embodiments where the individualhas cancer, these include direct measurements of tumor size viapalpation or visual observation, indirect measurement of tumor size byx-ray or other imaging techniques; an improvement as assessed by directtumor biopsy and microscopic examination of the tumor sample; themeasurement of an indirect tumor marker (e.g., PSA for prostate cancer)or an antigen identified according to the methods described herein, adecrease in pain or paralysis; improved speech, vision, breathing orother disability associated with the tumor; increased appetite; or anincrease in quality of life as measured by accepted tests orprolongation of survival. It will be apparent to one of skill in the artthat the dosage will vary depending on the individual, the type ofneoplastic condition, the stage of neoplastic condition, whether theneoplastic condition has begun to metastasize to other location in theindividual, and the past and concurrent treatments being used.

C. Combination Therapies

Combination therapies may be particularly useful in decreasing orinhibiting unwanted neoplastic cell proliferation, decreasing theoccurrence of cancer, decreasing or preventing the recurrence of cancer,or decreasing or preventing the spread or metastasis of cancer. In suchcases the modulators of the instant invention may function assensitizing or chemosensitizing agents by removing the CSCs that wouldotherwise prop up and perpetuate the tumor mass and thereby allow formore effective use of current standard of care debulking or anti-canceragents. That is, the disclosed modulators may, in certain embodimentsprovide an enhanced effect (e.g., additive or synergistic in nature)that potentiates the mode of action of another administered therapeuticagent. In the context of the instant invention “combination therapy”shall be interpreted broadly and merely refers to the administration ofa modulator and one or more anti-cancer agents that include, but are notlimited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents,debulking agents, chemotherapeutic agents, radiotherapy andradiotherapeutic agents, targeted anti-cancer agents (including bothmonoclonal antibodies and small molecule entities), BRMs, therapeuticantibodies, cancer vaccines, cytokines, hormone therapies, radiationtherapy and anti-metastatic agents and immunotherapeutic agents,including both specific and nonspecific approaches.

There is no requirement for the combined results to be additive of theeffects observed when each treatment (e.g., antibody and anti-canceragent) is conducted separately. Although at least additive effects aregenerally desirable, any increased anti-tumor effect above one of thesingle therapies is beneficial. Furthermore, the invention does notrequire the combined treatment to exhibit synergistic effects. However,those skilled in the art will appreciate that with certain selectedcombinations that comprise preferred embodiments, synergism may beobserved.

In practicing combination therapy, the modulator and anti-cancer agentmay be administered to the subject simultaneously, either in a singlecomposition, or as two or more distinct compositions using the same ordifferent administration routes. Alternatively, the modulator mayprecede, or follow, the anti-cancer agent treatment by, e.g., intervalsranging from minutes to weeks. The time period between each delivery issuch that the anti-cancer agent and modulator are able to exert acombined effect on the tumor. In at least one embodiment, both theanti-cancer agent and the modulator are administered within about 5minutes to about two weeks of each other. In yet other embodiments,several days (2, 3, 4, 5, 6 or 7), several weeks (1, 2, 3, 4, 5, 6, 7 or8) or several months (1, 2, 3, 4, 5, 6, 7 or 8) may lapse betweenadministration of the modulator and the anti-cancer agent.

The combination therapy may be administered once, twice or at least fora period of time until the condition is treated, palliated or cured. Insome embodiments, the combination therapy is administered multipletimes, for example, from three times daily to once every six months. Theadministering may be on a schedule such as three times daily, twicedaily, once daily, once every two days, once every three days, onceweekly, once every two weeks, once every month, once every two months,once every three months, once every six months or may be administeredcontinuously via a minipump. The combination therapy may be administeredvia any route, as noted previously. The combination therapy may beadministered at a site distant from the site of the tumor.

In one embodiment a modulator is administered in combination with one ormore anti-cancer agents for a short treatment cycle to a subject in needthereof. The invention also contemplates discontinuous administration ordaily doses divided into several partial administrations. The modulatorand anti-cancer agent may be administered interchangeably, on alternatedays or weeks; or a sequence of antibody treatments may be given,followed by one or more treatments of anti-cancer agent therapy. In anyevent, as will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics.

In another preferred embodiment the DLL3 modulators of the instantinvention may be used in maintenance therapy to reduce or eliminate thechance of tumor recurrence following the initial presentation of thedisease. Preferably the disorder will have been treated and the initialtumor mass eliminated, reduced or otherwise ameliorated so the patientis asymptomatic or in remission. At such time the subject may beadministered pharmaceutically effective amounts of the disclosedmodulators one or more times even though there is little or noindication of disease using standard diagnostic procedures. In someembodiments, the modulators will be administered on a regular scheduleover a period of time, such as weekly, every two weeks, monthly, everysix weeks, every two months, every three months every six months orannually. Given the teachings herein, one skilled in the art couldreadily determine favorable dosages and dosing regimens to reduce thepotential of disease recurrence. Moreover such treatments could becontinued for a period of weeks, months, years or even indefinitelydepending on the patient response and clinical and diagnosticparameters.

In yet another preferred embodiment the modulators of the presentinvention may be used to prophylactically or as an adjuvant therapy toprevent or reduce the possibility of tumor metastasis following adebulking procedure. As used in the instant disclosure a “debulkingprocedure” is defined broadly and shall mean any procedure, technique ormethod that eliminates, reduces, treats or ameliorates a tumor or tumorproliferation. Exemplary debulking procedures include, but are notlimited to, surgery, radiation treatments (i.e., beam radiation),chemotherapy, immunotherapy or ablation. At appropriate times readilydetermined by one skilled in the art in view of the instant disclosurethe disclosed modulators may be administered as suggested by clinical,diagnostic or theragnostic procedures to reduce tumor metastasis. Themodulators may be administered one or more times at pharmaceuticallyeffective dosages as determined using standard techniques. Preferablythe dosing regimen will be accompanied by appropriate diagnostic ormonitoring techniques that allow it to be modified.

Yet other embodiments of the invention comprise administering thedisclosed modulators to subjects that are asymptomatic but at risk ofdeveloping a proliferative disorder. That is, the modulators of theinstant invention may be used in a truly preventative sense and given topatients that have been examined or tested and have one or more notedrisk factors (e.g., genomic indications, family history, in vivo or invitro test results, etc.) but have not developed neoplasia. In suchcases those skilled in the art would be able to determine an effectivedosing regimen through empirical observation or through acceptedclinical practices.

D. Anti-Cancer Agents

The term “anti-cancer agent” or “antiproliferative agent” means anyagent that can be used to treat a cell proliferative disorder such ascancer, and includes, but is not limited to, cytotoxic agents,cytostatic agents, anti-angiogenic agents, debulking agents,chemotherapeutic agents, radiotherapy and radiotherapeutic agents,targeted anti-cancer agents, BRMs, therapeutic antibodies, cancervaccines, cytokines, hormone therapies, radiation therapy andanti-metastatic agents and immunotherapeutic agents. It will beappreciated that, in selected embodiments as discussed above, suchanti-cancer agents may comprise conjugates and may be associated withmodulators prior to administration. In certain embodiments the disclosedanti-cancer agent will be linked to a DLL3 modulator to provide an ADCas set forth herein.

As used herein the term “cytotoxic agent” means a substance that istoxic to the cells and decreases or inhibits the function of cellsand/or causes destruction of cells. Typically, the substance is anaturally occurring molecule derived from a living organism. Examples ofcytotoxic agents include, but are not limited to, small molecule toxinsor enzymatically active toxins of bacteria (e.g., Diptheria toxin,Pseudomonas endotoxin and exotoxin, Staphylococcal enterotoxin A),fungal (e.g., α-sarcin, restrictocin), plants (e.g., abrin, ricin,modeccin, viscumin, pokeweed anti-viral protein, saporin, gelonin,momoridin, trichosanthin, barley toxin, Aleurites fordii proteins,dianthin proteins, Phytolacca mericana proteins (PAPI, PAPII, andPAP-S), Momordica charantia inhibitor, curcin, crotin, saponariaofficinalis inhibitor, gelonin, mitegellin, restrictocin, phenomycin,neomycin, and the tricothecenes) or animals, (e.g., cytotoxic RNascs,such as extracellular pancreatic RNases; DNase I, including fragmentsand/or variants thereof).

For the purposes of the instant invention a “chemotherapeutic agent”comprises a chemical compound that non-specifically decreases orinhibits the growth, proliferation, and/or survival of cancer cells(e.g., cytotoxic or cytostatic agents). Such chemical agents are oftendirected to intracellular processes necessary for cell growth ordivision, and are thus particularly effective against cancerous cells,which generally grow and divide rapidly. For example, vincristinedepolymerizes microtubules, and thus inhibits cells from enteringmitosis. In general, chemotherapeutic agents can include any chemicalagent that inhibits, or is designed to inhibit, a cancerous cell or acell likely to become cancerous or generate tumorigenic progeny (e.g.,TIC). Such agents are often administered, and are often most effective.In combination, e.g., in regimens such as CHOP or FOLFIRI. Again, inselected embodiments such chemotherapeutic agents may be conjugated tothe disclosed modulators.

Examples of anti-cancer agents that may be used in combination with (orconjugated to) the modulators of the present invention include, but arenot limited to, alkylating agents, alkyl sulfonates, aziridines,ethylenimines and methylamelamines, acetogenins, a camptothecin,bryostatin, callystatin, CC-1065, cryptophycins, dolastatin,duocarmycin, eleutherobin, pancratistatin, a sarcodictyin, spongistatin,nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin,hisphosphonates, esperamicin, chromoprotein enediyne antiohioticchromophores, aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, caciinomycin, carabicin, carminomycin, carzinophilin,chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin, epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid,nogalaniycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tuhercidin,ubenimex, zinostatin, zorubicin; anti-metabolites, erlotinib,vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acidanalogues, purine analogs, androgens, anti-adrenals, folic acidreplenisher such as frolinic acid, aceglatone, aldophosphamideglycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil,bisantrene, edatraxate, defofamine, demecolcine, diaziquone,elfornithine, elliptinium acetate, an epothilone, etoglucid, galliumnitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone,mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet,pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide,procarbazinc, PSK® polysaccharide complex (JHS Natural Products, Eugene,Oreg.), razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine;methotrexate; platinum analogs, vinblastine; platinum; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine;novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda;ibandronate; irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS2000; difluorometihylornithine; retinoids; capecitabine; combretastatin;leucovorin; oxaliplatin; inhibitors of PKC-alpha, Raf, H-Ras, EGFR andVEGF-A that reduce cell proliferation and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens and selective estrogenreceptor modulators, aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,and anti-androgens; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, ribozymes such as a VEGFexpression inhibitor and a HER2 expression inhibitor; vaccines,PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH;Vinorelbine and Esperamicins and pharmaceutically acceptable salts,acids or derivatives of any of the above.

In other embodiments the modulators of the instant invention may be usedin combination with any one of a number of antibodies (orimmunotherapeutic agents) presently in clinical trials or commerciallyavailable. To this end the disclosed modulators may be used incombination with an antibody selected from the group consisting ofabagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab,amatuxitmab, anattimomab, arcitumomab, bavituximab, bectumomab,bevacizumab, bivatuzumab, blinatumomb, brentuximab, cantuzumab,catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab,conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab,detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab,ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab,figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab,girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab,indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab,labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab,mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab,moxetumomab, narnatumab, naptumomab, necitumumab, nimotuzumab,nofetumomabn, ocaratuzumab, ofatumumab, olaratumab, onartuzumab,oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab,pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab,rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab,siltuximab, simtuzumab, solitomab, tacatuzumab, taplitumomab,tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab,tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab,zalutumumab, CC49, 3F8 and combinations thereof.

Still other particularly preferred embodiments will comprise the use ofantibodies approved for cancer therapy including, but not limited to,rituximab, trastuzumab, gemtuzumab ozogamcin, alemtuzumab, ibritumomabtiuxetan, tositumomab, bevacizumab, cetuximab, panitumumab, ofatumumab,ipilimumab and brentuximab vedotin. Those skilled in the art will beable to readily identify additional anti-cancer agents that arecompatible with the teachings herein.

E. Radiotherapy

The present invention also provides for the combination of modulatorswith radiotherapy (i.e., any mechanism for inducing DNA damage locallywithin tumor cells such as gamma-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions and the like). Combination therapyusing the directed delivery of radioisotopes to tumor cells is alsocontemplated, and may be used in connection with a targeted anti-canceragent or other targeting means. Typically, radiation therapy isadministered in pulses over a period of time from about 1 to about 2weeks. The radiation therapy may be administered to subjects having headand neck cancer for about 6 to 7 weeks. Optionally, the radiationtherapy may be administered as a single dose or as multiple, sequentialdoses.

XI. Indications

It will be appreciated that the modulators of the instant invention maybe used to diagnose, treat or inhibit the occurrence or recurrence ofany DLL3 associated disorder. Accordingly, whether administered alone orin combination with an anti-cancer agent or radiotherapy, the modulatorsof the invention are particularly useful for generally treatingneoplastic conditions in patients or subjects which may include benignor malignant tumors (e.g., adrenal, liver, kidney, bladder, breast,gastric, ovarian, colorectal, prostate, pancreatic, lung, thyroid,hepatic, cervical, endometrial, esophageal and uterine carcinomas;sarcomas; glioblastomas; and various head and neck tumors); leukemiasand lymphoid malignancies; other disorders such as neuronal, glial,astrocytal, hypothalamic and other glandular, macrophagal, epithelial,stromal and blastocoelic disorders; and inflammatory, angiogenic,immunologic disorders and disorders caused by pathogens. Particularly,key targets for treatment are ncoplastic conditions comprising solidtumors, although hematologic malignancies are within the scope of theinvention. Preferably the “subject” or “patient” to be treated will behuman although, as used herein, the terms are expressly held to compriseany mammalian species.

More specifically, neoplastic conditions subject to treatment inaccordance with the instant invention may be selected from the groupincluding, but not limited to, adrenal gland tumors, AIDS-associatedcancers, alveolar soft part sarcoma, astrocytic tumors, bladder cancer(squamous cell carcinoma and transitional cell carcinoma), bone cancer(adamantinoma, aneurismal bone cysts, osteochondroma, osteosarcoma),brain and spinal cord cancers, metastatic brain tumors, breast cancer,carotid body tumors, cervical cancer, chondrosarcoma, chordoma,chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer,colorectal cancer, cutaneous benign fibrous histiocytomas, desmoplasticsmall round cell tumors, ependymomas, Ewing's tumors, extraskeletalmyxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasiaof the bone, gallbladder and bile duct cancers, gestationaltrophoblastic disease, germ cell tumors, head and neck cancers, isletcell tumors, Kaposi's Sarcoma, kidney cancer (nephroblastoma, papillaryrenal cell carcinoma), leukemias, lipoma/benign lipomatous tumors,liposarcoma/malignant lipomatous tumors, liver cancer (hepatoblastoma,hepatocellular carcinoma), lymphomas, lung cancers (small cellcarcinoma, adenocarcinoma, squamous cell carcinoma, large cell carcinomaetc.), medulloblastoma, melanoma, meningiomas, multiple endocrineneoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma,neuroendocrine tumors, ovarian cancer, pancreatic cancers, papillarythyroid carcinomas, parathyroid tumors, pediatric cancers, peripheralnerve sheath tumors, phaeochromocytoma, pituitary tumors, prostatecancer, posterious unveal melanoma, rare hematologic disorders, renalmetastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcomas, skincancer, soft-tissue sarcomas, squamous cell cancer, stomach cancer,synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, thyroidmetastatic cancer, and uterine cancers (carcinoma of the cervix,endometrial carcinoma, and lelomyoma).

In certain preferred embodiments the proliferative disorder willcomprise a solid tumor including, but not limited to, adrenal, liver,kidney, bladder, breast, gastric, ovarian, cervical, uterine,esophageal, colorectal, prostate, pancreatic, lung (both small cell andnon-small cell), thyroid, carcinomas, sarcomas, glioblastomas andvarious head and neck tumors. In other preferred embodiments, and asshown in the Examples below, the disclosed modulators are especiallyeffective at treating small cell lung cancer (SCLC) and non-small celllung cancer (NSCLC) (e.g., squamous cell non-small cell lung cancer orsquamous cell small cell lung cancer). In one embodiment, the lungcancer is refractory, relapsed or resistant to a platinum based agent(e.g., carboplatin, cisplatin, oxaliplatin, topotecan) and/or a taxane(e.g., docetaxel, paclitaxel, larotaxel or cabazitaxel). Further, inparticularly preferred embodiments the disclosed modulators may be usedin a conjugated form to treat small cell lung cancer.

With regard to small cell lung cancer particularly preferred embodimentswill comprise the administration of conjugated modulators (ADCs). Inselected embodiments the conjugated modulators will be administered topatients exhibiting limited stage disease. In other embodiments thedisclosed modulators will be administered to patients exhibitingextensive stage disease. In other preferred embodiments the disclosedconjugated modulators will be administered to refractory patients (i.e.,those who recur during or shortly after completing a course of initialtherapy). Still other embodiments comprise the administration of thedisclosed modulators to sensitive patients (i.e, those whose relapse islonger than 2-3 months after primary therapy. In each case it will beappreciated that compatible modulators may be in a conjugated orunconjugated state depending the selected dosing regimen and theclinical diagnosis.

As discussed above the disclosed modulators may further be used toprevent, treat or diagnose tumors with neuroendocrine features orphenotypes including neuroendocrine tumors. True or canonicalneuroendocrine tumors (NETs) arising from the dispersed endocrine systemare relatively rare, with an incidence of 2-5 per 100,000 people, buthighly aggressive. Neuroendocrine tumors occur in the kidney,genitourinary tract (bladder, prostate, ovary, cervix, and endometrium),gastrointestinal tract (colon, stomach), thyroid (medullary thyroidcancer), and lung (small cell lung carcinoma and large cellneuroendocrine carcinoma). These tumors may secrete several hormonesincluding serotonin and/or chromogranin A that can cause debilitatingsymptoms known as carcinoid syndrome. Such tumors can be denoted bypositive immunohistochemical markers such as neuron-specific enolase(NSE, also known as gamma enolase, gene symbol=ENO2), CD56 (or NCAM1),chromogranin A (CHGA), and synaptophysin (SYP) or by genes known toexhibit elevated expression such as ASCL1. Unfortunately traditionalchemotherapies have not been particularly effective in treating NETs andliver metastasis is a common outcome.

While the disclosed modulators may be advantageously used to treatneuroendocrine tumors they may also be used to treat, prevent ordiagnose pseudo neuroendocrine tumors (pNETs) that genotypically orphenotypically mimic, resemble or exhibit common traits with canonicalneuroendocrine tumors. Pseudo neuroendocrine tumors or tumors withneuroendocrine features are tumors that arise from cells of the diffuseneuroendocrine system or from cells in which a neuroendocrinedifferentiation cascade has been aberrantly reactivated during theoncogenic process. Such pNETs commonly share certain phenotypic orbiochemical characteristics with traditionally defined neuroendocrinetumors, including the ability to produce subsets of biologically activeamines, neurotransmitters, and peptide hormones. Histologically, suchtumors (NETs and pNETs) share a common appearance often showing denselyconnected small cells with minimal cytoplasm of bland cytopathology andround to oval stippled nuclei. For the purposes of the instant inventioncommonly expressed histological markers or genetic markers that may beused to define neuroendocrine and pseudo neuroendocrine tumors include,but are not limited to, chromogranin A, CD56, synaptophysin, PGP9.S,ASCL1 and neuron-specific enolase (NSE).

Accordingly the modulators of the instant invention may beneficially beused to treat both pseudo neuroendocrine tumors and canonicalneuroendocrine tumors. In this regard the modulators may be used asdescribed herein to treat neuroendocrine tumors (both NET and pNET)arising in the kidney, genitourinary tract (bladder, prostate, ovary,cervix, and endometrium), gastrointestinal tract (colon, stomach),thyroid (medullary thyroid cancer), and lung (small cell lung carcinomaand large cell neuroendocrine carcinoma). Moreover, the modulators ofthe instant invention may be used to treat tumors expressing one or moremarkers selected from the group consisting of NSE, CD56, synaptophysin,chromogranin A, ASCL1 and PGP9.5 (UCHL1). That is, the present inventionmay be used to treat a subject suffering from a tumor that is NSE⁺ orCD56⁺ or PGP9.5⁺ or ASCL1⁺ or SYP⁺ or CHGA⁺ or some combination thereof.

With regard to hematologic malignancies it will be further beappreciated that the compounds and methods of the present invention maybe particularly effective in treating a variety of B-cell lymphomas,including low grade/NHL follicular cell lymphoma (FCC), mantle celllymphoma (MCL), diffuse large cell lymphoma (DLCL), small lymphocytic(SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuseNHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, highgrade small non-cleaved cell NHL, bulky disease NHL, Waldenstrom'sMacroglobulinemia, lymphoplasmacytoid lymphoma (LPL), mantle celllymphoma (MCL), follicular lymphoma (FL), diffuse large cell lymphoma(DLCL), Burkitt's lymphoma (BL), AIDS-related lymphomas, monocytic Bcell lymphoma, angioimmunoblastic lymphoadenopathy, small lymphocytic,follicular, diffuse large cell, diffuse small cleaved cell, large cellimmunoblastic lymphoblastoma, small, non-cleaved, Burkitt's andnon-Burkitt's, follicular, predominantly large cell; follicular,predominantly small cleaved cell; and follicular, mixed small cleavedand large cell lymphomas. See, Gaidono et al., “Lymphomas”, IN CANCER:PRINCIPLES & PRACTICE OF ONCOLOGY, Vol. 2: 2131-2145 (DeVita et al.,eds., 5.sup.th ed. 1997). It should be clear to those of skill in theart that these lymphomas will often have different names due to changingsystems of classification, and that patients having lymphomas classifiedunder different names may also benefit from the combined therapeuticregimens of the present invention.

The present invention also provides for a preventative or prophylactictreatment of subjects who present with benign or precancerous tumors.Beyond being a DLL3 associated disorder it is not believed that anyparticular type of tumor or proliferative disorder should be excludedfrom treatment using the present invention. However, the type of tumorcells may be relevant to the use of the invention in combination withsecondary therapeutic agents, particularly chemotherapeutic agents andtargeted anti-cancer agents.

XII. Articles of Manufacture

Pharmaceutical packs and kits comprising one or more containers,comprising one or more doses of a DLL3 modulator are also provided. Incertain embodiments, a unit dosage is provided wherein the unit dosagecontains a predetermined amount of a composition comprising, forexample, an anti-DLL3 antibody, with or without one or more additionalagents. For other embodiments, such a unit dosage is supplied insingle-use prefilled syringe for injection. In still other embodiments,the composition contained in the unit dosage may comprise saline,sucrose, or the like; a buffer, such as phosphate, or the like; and/orbe formulated within a stable and effective pH range. Alternatively, incertain embodiments, the composition may be provided as a lyophilizedpowder that may be reconstituted upon addition of an appropriate liquid,for example, sterile water. In certain preferred embodiments, thecomposition comprises one or more substances that inhibit proteinaggregation, including, but not limited to, sucrose and arginine. Anylabel on, or associated with, the container(s) indicates that theenclosed composition is used for diagnosing or treating the diseasecondition of choice.

The present invention also provides kits for producing single-dose ormulti-dose administration units of a DLL3 modulator and, optionally, oneor more anti-cancer agents. The kit comprises a container and a label orpackage insert on or associated with the container. Suitable containersinclude, for example, bottles, vials, syringes, etc. The containers maybe formed from a variety of materials such as glass or plastic andcontain a pharmaceutically effective amount of the disclosed modulatorsin a conjugated or unconjugated form. In other preferred embodiments thecontainer(s) comprise a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). Such kits will generally contain in asuitable container a pharmaceutically acceptable formulation of the DLL3modulator and, optionally, one or more anti-cancer agents in the same ordifferent containers. The kits may also contain other pharmaceuticallyacceptable formulations, either for diagnosis or combined therapy. Forexample, in addition to the DLL3 modulator of the invention such kitsmay contain any one or more of a range of anti-cancer agents such aschemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents;anti-metastatic agents; targeted anti-cancer agents; cytotoxic agents;and/or other anti-cancer agents. Such kits may also provide appropriatereagents to conjugate the DLL3 modulator with an anti-cancer agent ordiagnostic agent (e.g., see U.S. Pat. No. 7,422,739 which isincorporated herein by reference in its entirety).

More specifically the kits may have a single container that contains theDLL3 modulator, with or without additional components, or they may havedistinct containers for each desired agent. Where combined therapeuticsare provided for conjugation, a single solution may be pre-mixed, eitherin a molar equivalent combination, or with one component in excess ofthe other. Alternatively, the DLL3 modulator and any optionalanti-cancer agent of the kit may be maintained separately withindistinct containers prior to administration to a patient. The kits mayalso comprise a second/third container means for containing a sterile,pharmaceutically acceptable buffer or other diluent such asbaeteriostatic water for injection (BWH), phosphate-buffered saline(PBS), Ringer's solution and dextrose solution.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is preferably an aqueous solution, with asterile aqueous solution being particularly preferred. However, thecomponents of the kit may be provided as dried powder(s). When reagentsor components are provided as a dry powder, the powder can bereconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container.

As indicated briefly above the kits may also contain a means by which toadminister the antibody and any optional components to an animal orpatient, e.g., one or more needles or syringes, or even an eye dropper,pipette, or other such like apparatus, from which the formulation may beinjected or introduced into the animal or applied to a diseased area ofthe body. The kits of the present invention will also typically includea means for containing the vials, or such like, and other component inclose confinement for commercial sale, such as, e.g., injection orblow-molded plastic containers into which the desired vials and otherapparatus are placed and retained. Any label or package insert indicatesthat the DLL3 modulator composition is used for treating cancer, forexample small cell lung cancer.

In other preferred embodiments the modulators of the instant inventionmay be used in conjunction with, or comprise, diagnostic or therapeuticdevices useful in the diagnosis or treatment of proliferative disorders.For example, in on preferred embodiment the compounds and compositionsof the instant invention may be combined with certain diagnostic devicesor instruments that may be used to detect, monitor, quantify or profilecells or marker compounds involved in the etiology or manifestation ofproliferative disorders. For selected embodiments the marker compoundsmay comprise NSF, CD56, synaptophysin, chromogranin A, and PGP9.5.

In particularly preferred embodiments the devices may be used to detect,monitor and/or quantify circulating tumor cells either in vivo or invitro (see, for example, WO 2012/0128801 which is incorporated herein byreference). In still other preferred embodiments, and as discussedabove, the circulating tumor cells may comprise cancer stem cells.

XIII. Research Reagents

Other preferred embodiments of the invention also exploit the propertiesof the disclosed modulators as an instrument useful for identifying,monitoring, isolating, sectioning or enriching populations orsubpopulations of tumor initiating cells through methods such as flowcytometry, fluorescent activated cell sorting (FACS), magnetic activatedcell sorting (MACS) or laser mediated sectioning. Those skilled in theart will appreciate that the modulators may be used in severalcompatible techniques for the characterization and manipulation of TICincluding cancer stem cells (e.g., see U.S. Ser. Nos. 12/686,359,12/669,136 and 12/757,649 each of which is incorporated herein byreference in its entirety).

XIV. Miscellaneous

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Morespecifically, as used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aprotein” includes a plurality of proteins; reference to “a cell”includes mixtures of cells, and the like. In addition, ranges providedin the specification and appended claims include both end points and allpoints between the end points. Therefore, a range of 2.0 to 3.0 includes2.0, 3.0, and all points between 2.0 and 3.0.

Generally, nomenclature used in connection with, and techniques of, celland tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Abbas et al., Cellular and Molecular Immunology,6^(th) ed., W.B. Saunders Company (2010); Sambrook J. & Russell D.Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlowand Lane Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al.,Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003).Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclature used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Moreover, anysection headings used herein are for organizational purposes only andare not to be construed as limiting the subject matter described.

XV. DLL3 References

All references or documents disclosed or cited within thisspecification, including those set forth immediately below are, withoutlimitation, incorporated herein by reference in their entirety.

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Notch in the development of thyroid C-cells    and the treatment of medullary thyroid cancer. Am J Transl Res.    2:119-25. PMID: 20182588.-   de la Pompa J L et al (1997). Conservation of the Notch signaling    pathway in mammalian neurogenesis. Development. 124:1139-48. PMID:    9102301.-   D'Souza B et al. (2010). Canonical and non-canonical Notch ligands.    Curr Top Dev Biol. 92:73-129. PMID: 20816393.-   Dunwoodie S L (2009). The role of Notch in patterning the human    vertebral column. Curr Opin Genet Dev. 19:329-37. PMID:19608404.-   Dutta S et al., (2008). Notch signaling regulates endocrine cell    specification in the zebrafish anterior pituitary. Dev Biol.    319:248-57. PubMed PMID: 18534570.-   Fre S et al. (2005). Notch signals control the fate of immature    progenitor cells in the intestine. Nature. 435:964-8. PMID:    15959516.-   Fre S et al. (2009). Notch and Wnt signals cooperatively control    cell proliferation and tumorigenesis in the intestine. Proc Natl    Acad Sci U S A. 106:6309-14. PMID: 19251639.-   Galluzzo P, and Bocchetta M (2011). Notch signaling in lung cancer.    Expert Rev Anticancer Ther. 11:533-40. PMID: 21504320.-   Geffers I et al. (2007). Divergent functions and distinct    localization of the Notch ligands DLL1 and DLL3 in vivo. J Cell    Biol. 178:465-76, PMID: 17664336.-   Glittenberg M, et al., (2006). Role of conserved intracellular    motifs in Serrate signalling, cis-inhibition and endocytosis.    EMBO J. 25:4697-706. PMID: 17006545.-   Goldbeter A, and Pourquié O (2008). Modeling the segmentation clock    as a network of coupled oscillations in the Notch, Wnt and FGF    signaling pathways. J Theor Biol. 252:574-85.PMID: 18308339.-   Habener J F et al. (2005). Minireview: transcriptional regulation in    pancreatic development. Endocrinology. 146:1025-34. PMID: 15604203.-   Harris P J el al. (2012). Targeting embryonic signaling pathways in    cancer therapy. Expert Opin Ther Targets. PMID: 22239436.-   Henke R M et al. (2009). Ascl1 and Neurog2 form novel complexes and    regulate Delta-like3 (DLL3) expression in the neural tube. Dev Biol.    328:529-40. PMID:19389376.-   Hoyne G F, et al. (2011). A cell autonomous role for the Notch    ligand Delta-like 3 in αβ T-cell development. Immunol Cell Biol.    89:696-705. PMID: 21151194.-   Huber K et al., (2002). Development of chromaffin cells depends on    MASH1 function. Development. 129:4729-38. PMID: 12361965.-   Ito T et al. (2000). Basic helix-loop-helix transcription factors    regulate the neuroendocrine differentiation of fetal mouse pulmonary    epithelium. Development. 127:3913-21. PMID: 10952889.-   Jensen J et al. (2000). Control of endodermal endocrine development    by Hes-1, Nat Genet. 24:36-44. PMID: 10615124.-   Kageyama R, et al. (2007). Oscillator mechanism of Notch pathway in    the segmentation clock. Dev Dyn. 236:1403-9. PMID:17366573.-   Kameda Y et al. (2007). Mash1 regulates the development of C cells    in mouse thyroid glands. Dev Dyn. 236:262-70. PMID: 17103415.-   Klein T, et al. (1997). An intrinsic dominant negative activity of    serrate that is modulated during wing development in Drosophila. Dev    Biol. 189:123-34. PMID: 9281342.-   Klimstra D S, et al. (2010). The pathologic classification of    neuroendocrine tumors: a review of nomenclature, grading, and    staging systems. Pancreas. 39:707-12. PMID: 20664470.-   Klöppel G. (2011). Classification and pathology of    gastroenteropancreatic neuroendocrine neoplasms. Endocr Relat    Cancer. 18 Suppl 1:51-16. PMID: 22005112.-   Koch U and Radtke F (2010). Notch signaling in solid tumors. Curr    Top Dev Biol. 92:411-55. PMID: 20816403.-   Kusumi K et al. (1998). The mouse pudgy mutation disrupts Delta    homologue DLL3 and initiation of early somite boundaries. Nat Genet.    19:274-8. PMID: 9662403.-   Ladi E et al. (2005). The divergent DSL ligand DLL3 does not    activate Notch signaling but cell autonomously attenuates signaling    induced by other DSL ligands. J Cell Biol. 170:983-92.    PMID:16144902.-   Liu J et al. (2010). Notch signaling in the regulation of stem cell    self-renewal and differentiation. Curr Top Dev Biol. 92:367-409.    PMID: 20816402.-   Nagase H et al. (2011). γ-Secretase-regulated signaling pathways,    such as notch signaling, mediate the differentiation of    hematopoictic stem cells, development of the immune system, and    peripheral immune responses. Curr Stem Cell Res Ther. 6:131-41.    PMID: 21190540.-   Raetzman L T et al. (2004). Developmental regulation of Notch    signaling genes in the embryonic pituitary: Prop1 deficiency affects    Notch2 expression. Dev Biol. 265:329-40. PMID: 14732396.-   Rebay I, et al., (1991). Specific EGF repeats of Notch mediate    interactions with Delta and Serrate: implications for Notch as a    multifunctional receptor. 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XVI. Selected Embodiments of the Invention

In addition to the disclosure and Examples herein, the present inventionis directed to selected embodiments specifically set forth immediatelybelow.

Putative Claims:

-   1. An isolated DLL3 modulator.-   2. The isolated DLL3 modulator of claim 1, wherein the DLL3    modulator comprises a DLL3 antagonist.-   3. The isolated DLL3 modulator of claim 1, wherein the DLL3    modulator comprises an antibody or immunoreactive fragment thereof.-   4. The isolated DLL3 modulator of claim 3 wherein the antibody or    immunoreactive fragment thereof comprises a monoclonal antibody.-   5. The isolated DLL3 modulator of claim 4 wherein the monoclonal    antibody is selected from the group consisting of chimeric    antibodies, humanized antibodies and human antibodies.-   6. The isolated DLL3 modulator of claim 4 wherein said monoclonal    antibody comprises a neutralizing antibody.-   7. The isolated DLL3 modulator of claim 4 wherein said monoclonal    antibody comprises a depleting antibody.-   8. The isolated DLL3 modulator of claim 4 wherein said monoclonal    antibody comprises an internalizing antibody.-   9. The isolated DLL3 modulator of claim 8 wherein said monoclonal    antibody further comprises a cytotoxic agent.-   10. The isolated DLL3 modulator of claim 4 wherein said monoclonal    antibody comprises a light chain variable region having three    complementarity determining regions and a heavy chain variable    region having three complemnentarity determining regions wherein the    heavy and light chain complementarity determining regions comprise    at least one complementarity determining region set forth in FIG.    11A and FIG. 11B.-   11. The isolated DLL3 modulator of claim 4 wherein said monoclonal    antibody comprises a light chain variable region and a heavy chain    variable region wherein said light chain variable region comprises    an amino acid sequence having at least 60% identity to an amino acid    sequence selected from the group consisting of amino acid sequences    as set forth in SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID    NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34,    SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID    NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52,    SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID    NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70,    SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78 SEQ ID    NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88,    SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID    NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO:    106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114,    SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ    ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID    NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:    140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148,    SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ    ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162 SEQ ID NO: 164, SEQ ID    NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:    174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182,    SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ    ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID    NO: 200 and SEQ ID NO: 202 and wherein said heavy chain variable    region comprises an amino acid sequence having at least 60% identity    to an amino acid sequence selected from the group consisting of    amino acid sequences as set forth in SEQ ID NO: 21, SEQ ID NO: 23,    SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID    NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,    SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID    NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,    SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID    NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,    SEQ ID NO: 79, SEQ ID NO: 81, SEQ 1D NO: 83, SEQ ID NO: 85, SEQ ID    NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95,    SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID    NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO:    113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121,    SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ    ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID    NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:    147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155,    SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ    ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID    NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO:    181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189,    SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ    ID NO: 199, SEQ ID NO: 201 and SEQ ID NO: 203.-   12. An isolated DLL3 modulator comprising a CDR from any one of the    heavy or light chain variable regions set forth in of claim 11.-   13. An isolated DLL3 modulator comprising a competing antibody    wherein said competing antibody inhibits the binding of an isolated    DLL3 modulator of claim 10 or 11 to DLL3 by at least about 40%.-   14. A nucleic acid encoding an amino acid heavy chain variable    region or an amino acid light chain variable region of claim 11.-   15. A vector comprising the nucleic acid of claim 14.-   16. The isolated DLL3 modulator of claim 1 comprising an amino acid    sequence as set forth in SEQ ID NO: 3 or a fragment thereof.-   17. The isolated DLL3 modulator of claim 16 wherein the DLL3    modulator further comprises at least a portion of an immunoglobulin    constant region.-   18. The isolated DLL3 modulator of claim 1 wherein said modulator    reduces the frequency of tumor initiating cells upon administration    to a subject in need thereof.-   19. The isolated DLL3 modulator of claim 18 wherein the reduction in    frequency is determined using flow cytometric analysis of tumor cell    surface markers known to enrich for tumor initiating cells.-   20. The isolated DLL3 modulator of claim 18 wherein the reduction in    frequency is determined using immunohistochemical detection of tumor    cell surface markers known to enrich for tumor initiating cells.-   21. The isolated DLL3 modulator of claim 18 wherein said tumor    initiating cells comprise tumor perpetuating cells.-   22. The isolated DLL3 modulator of claim 1 further comprising a    cytotoxic agent.-   23. A pharmaceutical composition comprising the isolated DLL3    modulator of claim 1.-   24. The pharmaceutical composition of claim 23 wherein said isolated    DLL3 modulator comprises a monoclonal antibody.-   25. The pharmaceutical composition of claim 24 wherein said    monoclonal antibody comprises a humanized antibody.-   26. The pharmaceutical composition of claim 25 wherein said    humanized antibody comprises a cytotoxic agent.-   27. The pharmaceutical composition of claim 26 wherein said    cytotoxic agent comprises a pyrrolobenzodiazepine.-   28. A method of treating a DLL3 associated disorder comprising    administering a therapeutically effective amount of a DLL3 modulator    to a subject in need thereof.-   29. The method of claim 28 wherein said DLL3 modulator comprises a    DLL3 antagonist.-   30. The method of claim 28 wherein said DLL3 modulator comprises an    antibody or immunoreactive fragment thereof.-   31. The method of claim 30 wherein the antibody or immunoreactive    fragment thereof comprises a monoclonal antibody.-   32. The method of claim 31 wherein the monoclonal antibody is    selected from the group consisting of chimeric antibodies, humanized    antibodies and human antibodies.-   33. The method of claim 32 wherein said monoclonal antibody    comprises a light chain variable region and a heavy chain variable    region wherein said light chain variable region comprises an amino    acid sequence having at least 60% identity to an amino acid sequence    selected from the group consisting of amino acid sequences as set    forth SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,    SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID    NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44,    SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID    NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,    SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID    NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78 SEQ ID NO: 80,    SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID    NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98,    SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ    ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID    NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:    124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132,    SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ    ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID    NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO:    158, SEQ ID NO: 160, SEQ ID NO: 162 SEQ ID NO: 164, SEQ ID NO: 166,    SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ    ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID    NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO:    192, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200    and SEQ ID NO: 202 and wherein said heavy chain variable region    comprises an amino acid sequence having at least 60% identity to an    amino acid sequence selected from the group consisting of amino acid    sequences as set forth in SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:    25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ    ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:    43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ    ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:    61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ    ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO:    79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ    ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO:    97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105,    SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ    ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID    NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:    131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139,    SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ    ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID    NO: 157, SEQ ID NO: 159, SEQ ID NO: 151, SEQ ID NO: 163, SEQ ID NO:    165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173,    SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ    ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID    NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO:    199, SEQ ID NO: 201 and SEQ ID NO: 203.-   34. The method of claim 33 wherein said monoclonal antibody is a    humanized antibody.-   35. The method of claim 31 wherein said monoclonal antibody    comprises a neutralizing antibody.-   36. The method of claim 31 wherein said monoclonal antibody    comprises an internalizing antibody.-   37. The method of claim 36 wherein said internalizing antibody    comprises a cytotoxic agent.-   38. The method of claim 37 wherein said cytotoxic agent comprises a    pyrrolobenzodiazepine.-   39. The method of claim 38 wherein said DLL3 associated disorder    comprises a neoplastic disorder.-   40. The method of claim 39 wherein said neoplastic disorder    comprises a tumor exhibiting neuroendocrine features.-   41. The method of claim 40 wherein said tumor exhibiting    neuroendocrine features comprises a neuroendocrine tumor.-   42. The method of claim 39 wherein said neoplastic disorder    comprises a hematologic malignancy.-   43. The method of claim 42 wherein said hematologic malignancy    comprises leukemia or lymphoma.-   44. The method of claim 39 wherein the subject suffering said    neoplastic disorder exhibits tumors comprising tumor initiating    cells.-   45. The method of claim 44 further comprising the step of reducing    the frequency of tumor initiating cells in said subject.-   46. The method of claim 45 wherein the reduction in frequency is    determined using flow cytometric analysis of tumor cell surface    markers known to enrich for tumor initiating cells or    immunohistochemical detection of tumor cell surface markers known to    enrich for tumor initiating cells.-   47. The method of claim 45 wherein the reduction in frequency is    determined using in vitro or in vivo limiting dilution analysis.-   48. The method of claim 47 wherein the reduction in frequency is    determined using in vivo limiting dilution analysis comprising    transplant of live human tumor cells into immunocompromised mice.-   49. The method of claim 48 wherein the reduction of frequency    determined using in vivo limiting dilution analysis comprises    quantification of tumor initiating cell frequency using Poisson    distribution statistics.-   50. The method of claim 47 wherein the reduction of frequency is    determined using in vitro limiting dilution analysis comprising    limiting dilution deposition of live human tumor cells into in vitro    colony supporting conditions.-   51. The method of claim 50 wherein the reduction of frequency    determined using in vitro limiting dilution analysis comprises    quantification of tumor initiating cell frequency using Poisson    distribution statistics.-   52. The method of claim 28 further comprising the step of    administering an anti-cancer agent.-   53. The method of claim 28 wherein said DLL3 modulator comprises one    or more CDRs from any one of SEQ ID NOS: 20 to 203.-   54. The method of claim 28 wherein said DLL3 modulator comprises a    pan-DLL modulator.-   55. A method of reducing the frequency of tumor initiating cells in    a subject in need thereof comprising the step of administering a    DLL3 modulator to said subject.-   56. The method of claim 55 wherein the tumor initiating cells    comprise tumor perpetuating cells.-   57. The method of claim 56 wherein said tumor perpetuating cells are    CD324⁺ or CD46⁺ cells.-   58. The method of claim 55 wherein said DLL3 modulator comprises an    antibody.-   59. The method of claim 58 wherein said antibody comprises a    monoclonal antibody.-   60. The method of claim 59 wherein said monoclonal antibody further    comprises a cytotoxic agent.-   61. The method of claim 55 wherein the subject is suffering from a    neoplastic disorder selected from the group consisting of adrenal    cancer, bladder cancer, cervical cancer, endometrial cancer, kidney    cancer, liver cancer, lung cancer, ovarian cancer, colorectal    cancer, pancreatic cancer, prostate cancer and breast cancer.-   62. The method of claim 55 wherein the frequency of tumor initiating    cells is reduced by at least 10%.-   63. The method of claim 55 wherein the reduction in frequency is    determined using flow cytometric analysis of tumor cell surface    markers known to enrich for tumor initiating cells or    immunohistochemical detection of tumor cell surface markers known to    enrich for tumor initiating cells.-   64. A method of treating a subject suffering from a hematologic    malignancy comprising the step of administering a DLL3 modulator to    said subject.-   65. The method of claim 64 wherein said DLL3 modulator comprises a    monoclonal antibody.-   66. A method of sensitizing a tumor in a subject for treatment with    an anti-cancer agent comprising the step of administering a DLL3    modulator to said subject.-   67. The method of claim 66 wherein said DLL3 modulator comprises an    antibody.-   68. The method of claim 66 wherein said tumor is a solid tumor.-   69. The method of claim 66 wherein said anti-cancer agent comprises    a chemotherapeutic agent.-   70. The method of claim 66 wherein said anti-cancer agent comprises    an immunotherapeutic agent.-   71. A method of diagnosing a proliferative disorder in a subject in    need thereof comprising the steps of:    -   a. obtaining a tissue sample from said subject;    -   b. contacting the tissue sample with at least one DLL3        modulator; and    -   c. detecting or quantifying the DLL3 modulator associated with        the sample.-   72. The method of claim 71 wherein the DLL3 modulator comprises a    monoclonal antibody.-   73. The method of claim 72 wherein the antibody is operably    associated with a reporter.-   74. An article of manufacture useful for diagnosing or treating DLL3    associated disorders comprising a receptacle comprising a DLL3    modulator and instructional materials for using said DLL3 modulator    to treat or diagnose the DLL3 associated disorder.-   75. The article of manufacture of claim 74 wherein said DLL3    modulator is a monoclonal antibody.-   76. The article of manufacture of claim 74 wherein the receptacle    comprises a readable plate.-   77. A method of treating a subject suffering from neoplastic    disorder comprising the step of administering a therapeutically    effective amount of at least one internalizing DLL3 modulator.-   78. The method of claim 77 wherein said DLL3 modulator comprises an    antibody.-   79. The method of claim 78 wherein said antibody comprises a    monoclonal antibody.-   80. The method of claim 79 wherein the monoclonal antibody further    comprises a cytotoxic agent.-   81. The method of claim 80 further comprising the step of    administering an anti-cancer agent.-   82. A method of treating a subject suffering from neoplastic    disorder comprising the step of administering a therapeutically    effective amount of at least one neutralizing DLL3 modulator.-   83. The method of claim 82 wherein said DLL3 modulator comprises an    antibody.-   84. The method of claim 83 wherein said antibody comprises a    monoclonal antibody.-   85. The method of claim 84 wherein said monoclonal antibody    comprises a humanized antibody.-   86. The method of claim 85 wherein said humanized antibody further    comprises a cytotoxic agent.-   87. The method of claim 82 wherein the neoplastic disorder comprises    a tumor exhibiting neuroendocrine features.-   88. A method of identifying, isolating, sectioning or enriching a    population of tumor initiating cells comprising the step of    contacting said tumor initiating cells with a DLL3 modulator.-   89. The method of claim 88 wherein said DLL3 modulator comprises an    antibody.-   90. A DLL3 modulator comprising a humanized antibody wherein said    humanized antibody comprises a light chain variable region and a    heavy chain variable region wherein said light chain variable region    comprises an amino acid sequence having at least 60% identity to an    amino acid sequence selected from the group consisting of amino acid    sequences as set forth in SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO:    208, SEQ ID NO: 210 and SEQ ID NO: 212 and wherein said heavy chain    variable region comprises an amino acid sequence having at least 60%    identity to an amino acid sequence selected from the group    consisting of amino acid sequences as set forth in SEQ ID NO: 205,    SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211 and SEQ ID NO: 213.-   91. A method inhibiting or preventing metastasis in a subject in    need thereof comprising the step of administering a pharmaceutically    effective amount of a DLL3 modulator.-   92. The method of claim 91 wherein the subject undergoes a debulking    procedure before or after the administration of the DLL3 modulator.-   93. The method of claim 92 wherein said debulking procedure    comprises the administration of at least one anti-cancer agent.-   94. A method of performing maintenance therapy on a subject in need    thereof comprising the step of administering a pharmaceutically    effective amount of a DLL3 modulator.-   95. The method of claim 94 wherein said subject was treated for a    neoplastic disorder prior to the administration of the DLL3    modulator.-   96. A method of depleting tumor initiating cells in a subject    suffering from a proliferative disorder comprising the step of    administering a DLL3 modulator.-   97. A method of diagnosing, detecting or monitoring a DLL3    associated disorder in vivo in a subject in need thereof comprising    the step of administering a DLL3 modulator.-   98. A method of diagnosing, detecting or monitoring a DLL3    associated disorder in a subject in need thereof comprising the step    contacting circulating tumor cells with a DLL3 modulator.-   99. The method of claim 98 wherein said contacting step occurs in    vivo.-   100. The method of claim 98 wherein said contacting step occurs in    vitro.-   101. A method of treating a tumor exhibiting neuroendocrine features    in a patient in need thereof comprising the step of administering a    therapeutically effective amount of a DLL3 modulator.-   102. The method of claim 101 wherein said tumor exhibiting    neuroendocrinc features is a neuroendocrine tumor.-   103. A DLL3 modulator derived from an antibody selected from the    group consisting of SC16.3, SC16.4, SC16.5, SC16.7, SC16.8, SC16.10,    SC16.11, SC16.13, SC16.15, SC16.18, SC16.19, SC16.20, SC16.21,    SC16.22, SC16.23, SC16.25, SC16.26, SC16.29, SC16.30, SC16.31,    SC16.34, SC16.35, SC16.36, SC16.38, SC16.39, SC16.41, SC16.42,    SC16.45, SC16.47, SC16.49, SC16.50, SC16.52, SC16.55, SC16.56,    SC16.57, SC16.58, SC16.61, SC16.62, SC16.63, SC16.65, SC16.67,    SC16.68, SC16.72, SC16.73, SC16.78, SC16.79, SC16.80, SC16.81,    SC16.84, SC16.88, SC16.101, SC16.103, SC16.104, SC16.105, SC16.106,    SC16.107, SC16.108, SC16.109, SC16.110, SC16.111, SC16.113,    SC16.114, SC16.115, SC16.116, SC16.117, SC16.118, SC16.120,    SC16.121, SC16.122, SC16.123, SC16.124, SC16.125, SC16.126,    SC16.129, SC16.130, SC16.131, SC16.132, SC16.133, SC16.134,    SC16.135, SC16.136, SC16.137, SC16.138, SC16.139, SC16.140,    SC16.141, SC16.142, SC16.143, SC16.144, SC16.147, SC16.148, SC16.149    and SC16.150.-   104. An isolated DLL3 modulator that binds to an epitope associated    with the EGF1 domain of DLL3.-   105. The DLL3 modulator of claim 104 wherein said DLL3 modulator    comprises an antibody or immunoreactive fragment thereof.-   106. The DLL3 modulator of claim 105 wherein said antibody or    immunoreactive fragment thereof comprises a monoclonal antibody.-   107. The DLL3 modulator of claim 106 wherein said DLL3 modulator    comprises an ADC.-   108. The DLL3 modulator of claim 107 wherein said ADC comprises a    pyrrolobenzodiazepine.-   109. The DLL3 modulator of claim 108 further comprising a linker.-   110. An isolated DLL3 modulator that binds to an epitope associated    with the EGF2 domain of DLL3.-   111. The DLL3 modulator of claim 110 wherein said DLL3 modulator    comprises an antibody or immunoreactive fragment thereof.-   112. The DLL3 modulator of claim 111 wherein said antibody or    immunoreactive fragment thereof comprises a monoclonal antibody.-   113. The DLL3 modulator of claim 112 wherein said DLL3 modulator    comprises an ADC.-   114. The DLL3 modulator of claim 113 wherein said ADC comprises a    pyrrolobenzodiazepine.-   115. The DLL3 modulator of claim 114 further comprising a linker.-   116. An isolated DLL3 modulator that binds to an epitope associated    with the EGF3 domain of DLL3.-   117. The DLL3 modulator of claim 116 wherein said DLL3 modulator    comprises an antibody or immunoreactive fragment thereof.-   118. The DLL3 modulator of claim 117 wherein said antibody or    immunoreactive fragment thereof comprises a monoclonal antibody.-   119. The DLL3 modulator of claim 118 wherein said DLL3 modulator    comprises an ADC.-   120. The DLL3 modulator of claim 119 wherein said ADC comprises a    pyrrolobenzodiazepine.-   121. The DLL3 modulator of claim 120 further comprising a linker.-   122. An isolated DLL3 modulator that binds to an epitope associated    with the EGF4 domain of DLL3.-   123. The DLL3 modulator of claim 122 wherein said DLL3 modulator    comprises an antibody or immunoreactive fragment thereof.-   124. The DLL3 modulator of claim 123 wherein said antibody or    immunoreactive fragment thereof comprises a monoclonal antibody.-   125. The DLL3 modulator of claim 124 wherein said DLL3 modulator    comprises an ADC.-   126. The DLL3 modulator of claim 125 wherein said ADC comprises a    pyrroiobenzodiazepine.-   127. The DLL3 modulator of claim 126 further comprising a linker.-   128. An isolated DLL3 modulator that binds to an epitope associated    with the EGF5 domain of DLL3.-   129. The DLL3 modulator of claim 128 wherein said DLL3 modulator    comprises an antibody or immunoreactive fragment thereof.-   130. The DLL3 modulator of claim 129 wherein said antibody or    immunoreactive fragment thereof comprises a monoclonal antibody.-   131. The DLL3 modulator of claim 130 wherein said DLL3 modulator    comprises an ADC.-   132. The DLL3 modulator of claim 131 wherein said ADC comprises a    pyrrolobeuzodiazepine.-   133. The DLL3 modulator of claim 132 further comprising a linker.-   134. An isolated DLL3 modulator that binds to an epitope associated    with the EGF6 domain of DLL3.-   135. The DLL3 modulator of claim 134 wherein said DLL3 modulator    comprises an antibody or immunoreactive fragment thereof.-   136. The DLL3 modulator of claim 135 wherein said antibody or    immunoreactive fragment thereof comprises a monoclonal antibody.-   137. The DLL3 modulator of claim 136 wherein said DLL3 modulator    comprises an ADC.-   138. The DLL3 modulator of claim 137 wherein said ADC comprises a    pyrrolobenzodiazepine.-   139. The DLL3 modulator of claim 138 further comprising a linker.-   140. An isolated DLL3 modulator that binds to an epitope associated    with the DSL domain of DLL3.-   141. The DLL3 modulator of claim 140 wherein said DLL3 modulator    comprises an antibody or immunoreactive fragment thereof.-   142. The DLL3 modulator of claim 141 wherein said antibody or    immunoreactive fragment thereof comprises a monoclonal antibody.-   143. The DLL3 modulator of claim 142 wherein said DLL3 modulator    comprises an ADC.-   144. The DLL3 modulator of claim 143 wherein said ADC comprises a    pyrrolobenzodiazepine.-   145. The DLL3 modulator of claim 144 further comprising a linker.-   146. An isolated DLL3 modulator that binds to an epitope associated    with the N-terminal domain of DLL3.-   147. The DLL3 modulator of claim 146 wherein said DLL3 modulator    comprises an antibody or immunoreactive fragment thereof.-   148. The DLL3 modulator of claim 147 wherein said antibody or    immunoreactive fragment thereof comprises a monoclonal antibody.-   149. The DLL3 modulator of claim 148 wherein said DLL3 modulator    comprises an ADC.-   150. The DLL3 modulator of claim 149 wherein said ADC comprises a    pyrrolobenzodiazepine.-   151. The DLL3 modulator of claim 150 further comprising a linker.-   152. An isolated DLL3 modulator residing in a bin selected from the    group consisting of bin A, bin B, bin C, bin D, bin E, bin F, bin G,    bin H and bin I.-   153. An isolated DLL3 modulator residing in a bin defined by a    reference antibody selected from the group consisting of SC16.3,    SC16.4, SC16.5, SC16.7, SC16.8, SC16.10, SC16.11, SC16.13, SC16.15,    SC16.18, SC16.19, SC16.20, SC16.21, SC16.22, SC16.23, SC16.25,    SC16.26, SC16.29, SC16.30, SC16.31, SC16.34, SC16.35, SC16.36,    SC16.38, SC16.39, SC16.41, SC16.42, SC16.45, SC16.47, SC16.49,    SC16.50, SC16.52, SC16.55, SC16.56, SC16.57, SC16.58, SC16.61,    SC16.62, SC16.63, SC16.65, SC16.67, SC16.68, SC16.72, SC16.73,    SC16.78, SC16.79, SC16.80, SC16.81., SC16.84, SC16.88, SC16.101,    SC16.103, SC16.104, SC16.105, SC16.106, SC16.107, SC16.108,    SC16.109, SC16.110, SC16.111, SC16.113, SC16.114, SC16.115,    SC16.116, SC16.117, SC16.118, SC16.120, SC16.121, SC16.122,    SC16.123, SC16.124, SC16.125, SC16.126, SC16.129, SC16.130,    SC16.131, SC16.132, SC16.133, SC16.134, SC16.135, SC16.136,    SC16.137, SC16.138, SC16.139, SC16.140, SC16.141, SC16.142,    SC16.143, SC16.144, SC16.147, SC16.148, SC16.149 and SC16.150.-   154. An antibody drug conjugate of the formula:    M-[L-D]n

or a pharmaceutically acceptable salt thereof wherein

-   -   a) M comprises a DLL3 modulator;    -   b) L comprises an optional linker;    -   c) D is a anti-proliferative agent; and    -   d) n is an integer from about 1 to about 20.

-   155. The antibody drug conjugate of claim 154 wherein said DLL3    modulator comprises an antibody or immunoreactive fragment thereof.

-   156. The antibody drug conjugate of claim 155 wherein said antibody    comprises a monoclonal antibody.

-   157. The antibody drug conjugate of claim 156 wherein said antibody    is derived from an antibody selected from the group consisting of    SC16.3, SC16.4, SC16.5, SC16.7, SC16.8, SC16.10, SC16.11, SC16.13,    SC16.15, SC16.18, SC16.19, SC16.20, SC16.21, SC16.22, SC16.23,    SC16.25, SC16.26, SC16.29, SC16.30, SC16.31, SC16.34, SC16.35,    SC16.36, SC16.38, SC16.39, SC16.41, SC16.42, SC16.45, SC16.47,    SC16.49, SC16.50, SC16.52, SC16.55, SC16.56, SC16.57, SC16.58,    SC16.61, SC16.62, SC16.63, SC16.65, SC16.67, SC16.68, SC16.72,    SC16.73, SC16.78, SC16.79, SC16.80, SC16.81, SC16.84, SC16.88,    SC16.101, SC16.103, SC16.104, SC16.105, SC16.106, SC16.107,    SC16.108, SC16.109, SC16.110, SC16.111, SC16.113, SC16.114,    SC16.115, SC16.116, SC16.117, SC16.118, SC16.120, SC16.121,    SC16.122, SC16.123, SC16.124, SC16.125, SC16.126, SC16.129,    SC16.130, SC16.131, SC16.132, SC16.133, SC16.134, SC16.135,    SC16.136, SC16.137, SC16.138, SC16.139, SC16.140, SC16.141,    SC16.142, SC16.143, SC16.144, SC16.147, SC16.148, SC16.149 and    SC16.150.

-   158. The antibody drug conjugate of claim 157 wherein said antibody    is humanized.

-   159. The antibody drug conjugate of claim 154 wherein the linker    comprises a cleavable linker.

-   160. The antibody drug conjugate of claim 159 wherein said cleavable    linker comprises a peptidyl linker.

-   161. The antibody drug conjugate of claim 154 wherein said    anti-proliferative agent comprises a cytotoxic agent.

-   162. The antibody drug conjugate of claim 161 wherein said cytotoxic    agent comprises a pyrrolobenzodiazepine.

-   163. The antibody drug conjugate of claim 162 wherein said    pyrrolobenzodiazepine comprises a pyrrolobenzodiazepine dimer.

-   164. A DLL3 modulator comprising a CDR from any one of SEQ ID NOS:    20-203.

-   165. The DLL3 modulator of claim 164 wherein said modulator    comprises a plurality of CDRs from any one of SEQ ID NOS: 20-203.

-   166. A DLL3 antibody modulator that competes for binding to a DLL3    protein with a reference antibody selected from the group consisting    of SC16.3, SC16.4, SC16.5, SC16.7, SC16.8, SC16.10, SC16.11,    SC16.13, SC16.15, SC16.18, SC16.19, SC16.20, SC16.21, SC16.22,    SC16.23, SC16.25, SC16.26, SC16.29, SC16.30, SC16.31, SC16.34,    SC16.35, SC16.36, SC16.38, SC16.39, SC16.41, SC16.42, SC16.45,    SC16.47, SC16.49, SC16.50, SC16.52, SC16.55, SC16.56, SC16.57,    SC16.58, SC16.61, SC16.62, SC16.63, SC16.65, SC16.67, SC16.68,    SC16.72, SC16.73, SC16.78, SC16.79, SC16.80, SC16.81, SC16.84,    SC16.88, SC16.101, SC16.103, SC16.104, SC16.105, SC16.106, SC16.107,    SC16.108, SC16.109, SC16.110, SC16.111, SC16.113, SC16.114,    SC16.115, SC16.116, SC16.117, SC16.118, SC16.120, SC16.121,    SC16.122, SC16.123, SC16.124, SC16.125, SC16.126, SC16.129,    SC16.130, SC16.131, SC16.132, SC16.133, SC16.134, SC16.135,    SC16.136, SC16.137, SC16.138, SC16.139, SC16.140, SC16.141,    SC16.142, SC16.143, SC16.144, SC16.147, SC16.148, SC16.149 and    SC16.150 wherein binding of the DLL3 antibody modulator to the DLL3    protein is inhibited by at least 30%.

-   167. A DLL3 modulator that binds to a DLL3 protein epitope    comprising amino acids Q93, P94, G95, A96 and P97 (SEQ ID NO: 9).

-   168. A DLL3 modulator that binds to a DLL3 protein epitope    comprising amino acids G203, R205 and P206 (SEQ ID NO: 10).

EXAMPLES

The present invention, thus generally described above, will beunderstood more readily by reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the instant invention. The examples are not intended to representthat the experiments below are all or the only experiments performed.Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

Example 1 Analysis of Marker Expression in Selected Tumors withNeuroendocrine Features

Neuroendocrine tumors (NETs) arising from the dispersed endocrine systemare rare, with an incidence of 2-5 per 100,000 people, but highlyaggressive. Neuroendocrine tumors occur in the adrenal gland, kidney,genitourinary tract (bladder, prostate, ovary, cervix, and endometrium),pancreas, gastrointestinal tract (stomach and colon), thyroid (medullarythyroid cancer), and lung (small cell lung carcinoma, large cellneuroendocrine carcinoma, and carcinoid). These tumors may secreteseveral hormones including serotonin and/or chromogranin A that cancause debilitating symptoms known as carcinoid syndrome. These tumorscan be denoted by positive immunohistochemical markers such asneuron-specific enolase (NSE, also known as gamma enolase, genesymbol=ENO2), CD56/NCAM1, and synaptophysin. Traditional chemotherapieshave not been successful in treating NETs, and mortality due tometastatic spread is a common outcome. Unfortunately, in most casessurgery is the only potential curative treatment, provided it takesplace following early detection and prior to tumor metastasis. In thiscontext work was undertaken to identify novel therapeutic targetsassociated with tumors comprising neuroendocrine features.

To identify and characterize such tumors as they exist in cancerpatients a large non-traditional xenograft (NTX) tumor bank wasdeveloped and maintained using art-recognized techniques. The NTX tumorbank, comprising a substantial number of discrete tumor cell lines, waspropagated in immunocompromised mice through multiple passages ofheterogeneous tumor cells originally obtained from numerous cancerpatients afflicted by a variety of solid tumor malignancies. (Note thatin some of the Examples and FIGS. herein the passage number of thetested sample is indicated by p0-p# appended to the sample designationwhere p0 is indicative of an unpassaged sample obtained directly from apatient tumor and p# is indicative of the number of times the tumor hasbeen passaged through a mouse prior to testing.) The continuedavailability of a large number of discrete early passage NTX tumor celllines having well defined lineages greatly facilitate the identificationand characterization of cells purified from the cell lines. In such workthe use of minimally passaged NTX cell lines simplifies in vivoexperimentation and provides readily verifiable results. Moreover, earlypassage NTX tumors respond to therapeutic agents such as irinotecan(i.e. Camptosar®) and Cisplatin/Etoposide regimens, which providesclinically relevant insights into underlying mechanisms driving tumorgrowth, resistance to current therapies and tumor recurrence.

As the NTX tumor cell lines were established, their phenotype wascharacterized in various ways to examine global gene expression. Toidentify which NTX lines in the bank might be NETs, gene expressionprofiles were generated by whole transcriptome sequencing and/ormicroarray analysis. Specifically, the data was examined to identifytumors expressing high levels of specific genes known to be elevated inNETs or used as histochemical markers of neuroendocrine differentiation(e.g., ASCL1, NCAM1, CHGA) as well as tumors with changes in NOTCHpathway genes indicative of suppression of NOTCH signaling (e.g.,reduced levels of NOTCH receptors, and changes to ligands and effectormolecules).

More particularly, upon establishing various NTX tumor cell lines as iscommonly done for human tumors in severely immune compromised mice, thetumors were resected after reaching 800-2,000 mm³ and the cells weredissociated and dispersed into suspension using art-recognized enzymaticdigestion techniques (see, for example, U.S.P.N. 2007/0292414 which isincorporated herein). The dissociated cell preparations from these NTXlines were then depleted of murine cells, and human tumor cellsubpopulations were then further isolated by fluorescence activated cellsorting and lysed in RLTplus RNA lysis buffer (Qiagen). These lysateswere then stored at −80° C. until used. Upon thawing, total RNA wasextracted using a RNeasy isolation kit (Qiagen) following the vendor'sinstructions and quantified on a Nanodrop spectrophotometer (ThermoScientific) and a Bloanalyzer 2100 (Agilent Technologies) again usingthe manufacturer's protocols and recommended instrument settings. Theresulting total RNA preparations were suitable for genetic sequencingand gene expression analysis.

Whole transcriptome sequencing using an Applied Biosystems (ABI) SOLID(Sequencing by Oligo Ligation/Detection) 4.5 or SOLiD 5500×l nextgeneration sequencing system (Life Technologies) was performed on RNAsamples from NTX lines. cDNA was generated from total RNA samples usingeither a modified whole transcriptome (WT) protocol from ABI designedfor low input total RNA or Ovation RNA-Seq System V2™ (NuGENTechnologies Inc.). The modified low input WT protocol uses 1.0 ng oftotal RNA to amplify mRNA at the 3′ end which leads to a heavy 3′ biasof mapped gene expression, while NuGen's system allows for a moreconsistent amplification throughout the transcript, and includesamplification of both mRNA and non-polyadenylated transcript cDNA usingrandom hexamers. The cDNA library was fragmented, and barcodes adapterswere added to allow pooling of fragment libraries from differentsamples.

ABI's SOLiD 4.5 and SOLiD 5500×l next generation sequencing platformsenables parallel sequencing of transcriptomes from multiple NTX linesand sorted populations. A cDNA library is constructed from each RNAsample, which is fragmented and barcoded. Barcodes on each fragmentlibrary allow multiple samples to be pooled at equal concentrations andrun together while ensuring sample specificity. The samples are takenthrough emulsion PCR using ABI's SOLiD™ EZ Bead™ robotics system, whichensures sample consistency. Paired-end sequencing generates a 50 baseread in the 5′ to 3′ direction and a 25 base read in the 3′ to 5′direction for each clonally amplified fragment on a single bead thatexists in the pool. In the case of the 5500×1 platform, for every set of8 samples pooled in the method mentioned above, beads are evenlydeposited into 6 single channel lanes on a single chip. This will, onaverage, generate more than 50 million 50 base reads and 50 million 25base reads for each of the 8 samples and generates a very accuraterepresentation of mRNA transcript level in the tumor cells. Datagenerated by the SOLiD platform mapped to 34,609 genes as annotated byRefSeq version 47 using NCBI version hgl9.2 of the published humangenome and provided verifiable measurements of RNA levels in mostsamples.

The SOLiD platform is able to capture not only expression, but SNPs,known and unknown alternative splicing events, small non-coding RNAs,and potentially new exon discoveries based solely on read coverage(reads mapped uniquely to previously un-annotated genomic locations).Thus, use of this next generation sequencing platform paired withproprietary data analysis and visualization software thus allowed fordiscovery of differential transcript expression as well as differencesand/or preferences for specific splice variants of expressed mRNAtranscripts. Sequencing data from the SOLiD platform is nominallyrepresented as a transcript expression value using the metrics RPM(reads per million) and RPKM (read per kilobase per million), enablingbasic differential expression analysis as is standard practice.

Whole transcriptome sequencing of four small cell lung cancer (SCLC)tumors (LU73, LU64, LU86 and LU95), one ovarian tumor (OV26) and a largecell neuroendocrine carcinoma (LCNEC; LU37) resulted in thedetermination of gene expression patterns commonly found in NETs (FIG.4A). More specifically, these tumors had high expression of several NETmarkers (ASCL1, NCAM1, CHGA) as well as reduced levels of Notchreceptors and effector molecules (e.g., HES1, HEY1) and elevated markersof Notch suppression (e.g., DLL3 and HES6). In contrast, 4 normal lungsamples, 3 lung adenocarcinoma tumors (LU137, LU146 and LU153), and 3squamous cell lung carcinomas (LU49, LU70 and LU76) all have expressionof various Notch receptors and effector molecules, and do not showelevated expression of Notch suppressors such as HES6 and DLL3.

After identifying which NTX in the tumor bank are NETs, each wasanalyzed using whole transcriptome sequencing data to find potentialtherapeutic targets upregulated in NETs when compared to non-NETs(including LU_SCC, LU_Ad, and normal lung). High expression of DLL3 wasfound in NET NTX tumors including SCLC, LCNEC, and OV26, compared to lowto non-existent expression in normal lung, normal ovary, other OV NTX,LU_Ad and LU_SCC NTX lines (FIG. 4B). High expression of DLL3 in NETsrelative to a variety of normal tissue types was of great interest, asDLL3 is a known suppressor of Notch signaling. Given this, and in viewof the generated data, DLL3 was selected for further analysis as apotential immunotherapeutic target.

With the discovery that DLL3 may prove to be a viable target formodulation and treatment of certain proliferative disorders, work wasundertaken to determine the expression pattern and levels of DLL3variants. As discussed above, there are two known splice variants ofDLL3 encoding proteins which differ only in that isoform 1 has anextended intracellular C-terminus (FIG. 1E). More specifically isoform 2is a 587 amino acid protein (FIG. 1D; SEQ ID NO: 4) encoded by mRNAvariant 2 (FIG. 1B; SEQ ID NO: 2), which contains exons 8a and 8c whileisoform 1 is a 618 amino acid protein (FIG. 1C; SEQ ID NO: 3) encoded bymRNA variant 1 (FIG. 1A; SEQ ID NO: 1), which contains exon 8b. Aschematic diagram illustrating the identical extracellular domain (ECD)of isoform 1 and isoform 2 in presented in FIG. 1F.

Again, using the whole transcriptome data obtained as described above,selected NET tumors were examined to determine the expression patternsof the aforementioned exons which, by extension, provides the expressionratio of the two isoforms. As shown in FIG. 5 it was found that whilethe particular expression ratio between the two isoforms may varysomewhat, isoform 1 expression was predominant in each tumor. In thisrespect it is important to note that, as described above, the cumulativeDLL3 expression (both isoforms) in each of the tested tumors waselevated with regard to normal tissues. Accordingly, while isoformratios may be indicative of certain tumor types and relevant togenotypic modulator selection it is not as critical with regard tophenotypic modulator strategies. That is, because the ECD region of bothDLL3 isoforms are identical, it is expected that a phenotypic modulatorof the instant invention directed to the ECD region (e.g., an anti-DLL3antibody) would react with either isoform. Thus it is the absoluteexpression levels of the DLL3 ECD (regardless of isoform) that isdispositive as to the effectiveness of such strategies.

Example 2 Microarray and RT-PCR Analysis of Gene Expression in SelectedNTX Tumors with Neuroendocrine Features

In an effort to identify additional NETs in the aforementioned NTX bankbeyond those for which SOLiD whole transcriptome data existed, a largerset of NTX lines was examined using microarray analysis. Specifically,2-6 μg of total RNA samples derived from whole tumors in 46 NTX lines orfrom 2 normal tissues were analyzed using a OneArray® microarrayplatform (Phalanx Biotech Group), which contains 29,187 probes designedagainst 19,380 genes in the human genome. More specifically, RNA sampleswere obtained (as described in Example 1) from forty-six patient derivedwhole NTX tumors comprising colorectal (CR), melanoma (SK), kidney (KD),lung (LU), ovarian (OV), endometrial (EM), breast (BR), liver (LIV), orpancreatic (PA) cancers. Normal colorectal (NormCR) and normal pancreas(NormPA) tissues were used as controls. Still more specifically, lungtumors were further subclassified as small cell lung cancers (SCLC),squamous cell cancers (SCC), or large cell neuroendocrine carcinoma(LCNEC). RNA samples were run in triplicate using the manufacturer'sprotocols and the resulting data was analyzed using standard industrypractices for normalizing and transforming the measured intensity valuesobtained for the subject gene in each sample. An unbiased PearsonSpearman hierarchical clustering algorithm in the R/BioConductor suiteof packages called hclust.2 was used to create a standard microarraydendrogram for these 48 samples. As known in the art R/BioConductor isan open-source, statistical programming language widely used inacademia, finance and the pharmaceutical industry for data analysis.Generally the tumors were arranged and clustered based on geneexpression patterns, expression intensity, etc.

As shown in FIG. 6A, the dendrogram derived from the 48 samples andacross all 19380 genes, clustered NTX lines together based upon theirtumor type or tissue of origin. Several tumors typically associated withneuroendocrine phenotypes clustered together on the branch denoted by(1); these included skin cancers, numerous lung cancers and other NETs.Interestingly, a sub-branch, denoted by (2), showed that two large celllung cancers with neuroendocrine features (LU50.LCNEC and LU37.LCNEC)and a small cell lung cancer (LU102.SCLC) clustered with an ovarian(OV26) and a kidney (KD66) tumor (cluster C) suggesting these latertumors also possessed neuroendocrine phenotypes. Moreover, FIG. 6A showscluster D which consists of 3 additional SCLC tumors, and to its rightis a small cluster containing an additional SCLC NIX (LU100) and aneuroendocrine endometrial tumor (EM6), all expected to possess someneuroendocrine features as is generally understood from the literatureand pathology experience in the clinic. The fact that cluster G,comprised of squamous cell carcinomas of the lung, can be found on acompletely different branch of the dendrogram of FIG. 6A indicates thatthe clustering is not driven exclusively by the organ of origin for theturmor.

Closer inspection of a collection of gene markers associated with NETs(FIG. 6B) shows that they are strongly expressed in tumors comprisingclusters C and D, while they are minimally expressed in tumors inCluster G (squamous cell carcinoma of the lung), suggesting clusters Cand D represent NETs or tumors with a neuroendocrine phenotype. Morespecifically, cluster C NETs highly express ASCL1, CALCA, CHGA, SST andNKX2-1, while cluster D NETs highly express CHGA, ENO2, and NCAM1, andit is the expression of these neuroendocrine phenotype genes that is inpart responsible for the clustering of these tumors. An interestingfeature is the strong expression of KIT in cluster D, a geneoccasionally reported to be associated with neuroendocrine tumors, butclearly linked to oncogenesis in other contexts. This is in contrast tothe SCC tumors in cluster G which lack strong expression any of thesegenes (FIG. 6B).

With regard to Notch signaling, tumors in cluster C show a phenotypeconsistent with a reduction in Notch signaling: a lack of expression ofany Notch receptor, a relative lack of JAG1 and HES1 expression, andstrong levels of ASCL1 expression (FIG. 6C). Interestingly, cluster Dshows high expression of HES6, a transcription factor that can supportASCL1 activity by antagonizing HES1 activity through heterodimerformation. Most importantly, these microarray data show high levels ofDLL3 transcription in tumors in clusters C and D (relative to clusterG), suggesting that in these tumor types, DLL3 provides an attractivetherapeutic target for treatment of NETs.

In view of the aforementioned results, mRNA expression of HES6 wasexamined from various NTX lines and normal tissues using an AppliedBiosystems 7900HT Machine (Life Technologies) to perform Taqmanreal-time quantitative RT-PCR (qRT-PCR) in accordance with themanufacturer's protocols. RNA was isolated as described above andchecked to ensure quality was suitable for gene expression analysis. RNAfrom normal tissues was purchased (Agilent Technologies and LifeTechnologies). 200 ng of RNA was used for cDNA synthesis using the cDNAarchive kit (Life Technologies). cDNA was used for qRT-PCR analysis onTaqman Low Density Arrays (TLDA; Life Technologies) which contained theHES6 Taqman assay to measure mRNA levels of HES6.

HES6 mRNA levels are shown for each NTX line or normal tissue sample(single dot on graph) after normalization to endogenous controls.Normalized values are plotted relative to the average expression in thenormal tissues of toxicity concern (NormTox). This technique allowed forthe rapid identification and characterization of a variety of tumorshaving neuroendocrine features from the NTX tumor bank throughmeasurement of HES6 and other relevant markers. FIG. 6D illustratesgeneral overexpression of HES6 in the sampled tumors with neuroendocrinefeatures (e.g., LU-SCLC, LU-LCNEC) compared to normal tissues, breast,colon, liver and other selected tumors. Significantly these microarrayand qPCR data show that at least some endometrial, kidney and ovariantumors may exhibit neuroendocrine tumor features (FIGS. 6A and 6D).

Example 3 RT-PCR Analysis of DLL3 in Tumors with Neuroendoerine Features

To confirm the generated SOLID and microarray data and extend theanalysis to additional NTX samples, DLL3 mRNA expression was analyzed byqRT-PCR using RNA samples from various NTX lines, primary biopsies andnormal tissues. The analysis was again performed using an AppliedBiosystems 7900HT Machine (Life Technologies) substantially as describedimmediately above but optimized for DLL3 detection. DLL3 expression isshown relative to the average expression in normal tissues andnormalized to expression of the endogenous control gene ALAS1. As seenin FIG. 7, qRT-PCR interrogation of gene expression showed that DLL3mRNA is elevated more than 10,000,000-fold in NET populations versusnormal tissues. In this Example the sampled tumors include additionalSCLC NTX lines beyond those tested previously as well as a number of RNAsamples derived from primary biopsies (p0). Taken together these datademonstrate that DLL3 gene expression is dramatically upregulated intumors exhibiting neuroendocrine features and, given that the samepattern is seen in primary biopsy samples, that the observedupregulation is not an artifact of growing human tumors in mice.

In addition, three subtypes of NSCLC as defined by clinical pathologyare also represented in FIG. 7: LU25 is a spindle cell lung carcinoma,LU50 is a large cell neuroendocrine carcinoma (LCNEC), and LU85 is asquamous cell carcinoma (SCC). The highest DLL3 expression was seen inthe LCNEC tumor LU50, though elevated levels were also noted in the SCCand spindle cell tumors. KDY66 and OV26, a kidney and ovarian tumor,respectively, clustered on the microarray with SCLC and LCNEC tumors(FIG. 6A), suggesting they comprise tumors exhibiting neuroendocrinefeatures (i.e., NETs or pNETs). Such a conclusion is corroborated by thehigh mRNA levels of DLL3 seen in both tumor samples (FIG. 7). While allof the tumors display a striking upregulation of DLL3 mRNA relative tonormal tissues (FIG. 7), comparison of tumors found both on FIGS. 6A and7 shows that subtle differences in measured DLL3 mRNA expression in FIG.7 correspond to differential clustering in FIG. 6A; e.g., cluster Ccontains KD66, LU50, OV26 and LU102, which are at the high end of DLL3expression as shown on FIG. 7, whereas LU85 and LU100, each of whichcluster away from clusters C and D in FIG. 6A, are among the lower endof DLL3 expression for the tumor samples measured. Small cell lungcancer tumors in cluster D in FIG. 6A (e.g., LU86, LU64, and LU95) showintermediate levels of DLL3 mRNA expression and may very well besusceptible to treatment with the modulators of the instant invention.

Example 4 Expression of DLL3 mRNA and Protein in Various Tumor Specimens

To extend the analysis of DLL3 expression to a wider array of tumorspecimens, Taqman qRT-PCR was performed substantially as described inthe previous Examples on a TissueScan™ qPCR (Origene Technologies)384-well array. This array enables comparison of gene expression across18 different solid tumor types, with multiple patient derived samplesfor each tumor type and from normal adjacent tissue.

In this regard, FIGS. 8A and 8B show the relative and absolute geneexpression levels, respectively, of DLL3 in whole tumor specimens (greydots) or normal adjacent tissue (NAT; white dots) from patients with oneof eighteen different solid tumor types. Data is normalized in FIG. 8Aagainst mean gene expression in NAT for each tumor type analyzed.Specimens in which DLL3 was not detected were assigned a Ct value of 50,which represents the last cycle of amplification in the experimentalprotocol. Each dot represents a single tissue specimen, with thegeometric mean value represented as a black line. Using this OrigeneTissueScan Array, overexpression of DLL3 was seen in a subset ofadrenal, breast, cervical, endometrial, lung, ovarian, pancreatic,thyroid and bladder cancer, many of which may represent NETs or tumorswith poorly differentiated neuroendocrine phenotypes. A subset of lungtumors showed the greatest overexpression of DLL3. The highestexpression was seen in 2 LCNEC tumors on the array. As shown by theabsolute gene expression in FIG. 8B, normal testis is the only normaltissue with high expression of DLL3. This suggests that DLL3 expressionin NETs and other tumorigenic cells might play a rote in tumorigenesisand/or tumor progression in a wide variety of tumors.

Given the elevated DLL3 transcript levels associated with varioustumors, work was undertaken to demonstrate a corresponding increase inthe expression of DLL3 protein in NETs relative to other tumors. To thisend a DLL3 sandwich ELISA was developed using the MSD Discovery Platform(Meso Scale Discovery, LLC) to detect and quantify DLL3 expression inselected NTX tumor samples. Briefly, NTX tumor samples were lysed andtotal protein concentration, as well as DLL3 protein concentration, weremeasured in the lysates using an electrochemiluminescence detectionbased sandwich ELISA format. More specifically, DLL3 concentrations fromthe samples were interpolated from electrochemoiluminescent values usinga standard curve generated from purified recombinant protein and areexpressed in FIG. 8C as nanograms of DLL3 per milligram of totalprotein.

More specifically NTX tumors were excised from mice and flash frozen ondry ice/ethanol. Protein Extraction Buffer (Biochain Institute, Inc.)was added to the thawed tumor pieces and tumors were pulverized using aTissue Lyser system (Qiagen). Lysates were cleared by centrifugation(20,000 g, 20 minutes, 4° C.) and protein was quantified usingbicinchoninic acid (BCA). Protein lysates were stored at −80° C. untilassayed.

MSD standard plates (Meso Scale Discovery, LLC) were coated overnight at4° C. with 30 μl of SC16.34 antibody (obtained as set forth in Example 7below) at 2 μg/ml in PBS. Plates were washed in PBST and blocked in 150ul MSD 3% Blocker A solution for 1 hour. Plates were again washed inPBST. 25 μl of the SC16.4 antibody (obtained as set forth in Example 7below) conjugated to the MSD sulfo-tag and was added to the washedplates at 0.5 μg/ml in MSD 1% Blocker A. 25 μl of 10× diluted lysate inMSD 1% Blocker A or serially diluted recombinant DLL3 standard in MSD 1%Blocker A containing 10% Protein Extraction Buffer was also added to thewells and incubated for 2 hours. Plates were washed in PBST. MSD ReadBuffer T with surfactant was diluted to 1× in water and 150 μl was addedto each well. Plates were read on a MSD Sector Imager 2400 using anintegrated software analysis program to derive DLL3 concentrations inNTX samples via interpolation. Values were then divided by total proteinconcentration to yield nanograms of DLL3 per milligram of total lysateprotein. The resulting concentrations are set forth in FIG. 8C whereineach spot represents concentrations derived from a single NTX tumorline. While each spot is derived from a single NTX line, in most casesmultiple biological samples were tested from the same NTX line andvalues were averaged to provide the data point.

In any event FIG. 8C shows that the highest expression of DLL3 was foundin SCLC, LCNEC, as well as other neuroendocrine tumors includingselected kidney samples and a single ovarian tumor. FIG. 8C alsodemonstrates that certain melanoma NTX lines exhibited elevated DLL3protein expression which is particularly interesting in that these NTXlines also clustered near NET NTX lines in the microarray analysisconducted in Example 4 (FIG. 6A).

These data, combined with the transcription data for DLL3 expression setforth above strongly reinforces the proposition that DLL3 determinantsprovide attractive targets for therapeutic intervention.

Example 5 Expression of NOTCH Receptors and Delta-like Ligands on theCell Surface of Selected NTX Tumor Lines

To further extend the observations from Examples 1 and 2 above, cellsisolated from several NTX tumors found in Clusters C and D (KDY66, OV26,LU64; FIG. 6A) as well as a SCLC tumor determined to have highexpression of DLL3 by SOLiD sequencing or qRT-PCR (LU73, FIGS. 4 and 7)were analyzed using flow cytometry for determination of the levels ofprotein expression for various Notch receptors and other DLL familymembers. Generally flow cytometry-based protein expression data wasgenerated using a FACSCanto II (BD Biosciences) as per themanufacturer's instructions. Data in FIG. 9 shows individual tumor cellsdisplayed as histogram plots, wherein the background staining of isotypecontrol antibodies is shown in the gray, filled histograms andexpression of the protein of interest, as determined using commerciallyavailable antibodies is displayed by the bold, black line.

As can be seen graphically in FIG. 9, little to no expression of any ofthe Notch receptors (e.g., NOTCH1-4) was observed in any of thesetumors, as determined relative to fluorescence minus one (FMO)isotype-control stained cells. This is indicated graphically by thehistograms, as well as numerically in the reported mean fluorescenceintensities (MFI) for each measurement. Similarly, the two lung cancerderived NTX cells showed no expression of either DLL1 or DLL4. Slightexpression of DLL4 alone (OV26) or DLL1 and DLL4 (KDY66) could beobserved for two of the tumors. In general, these observations confirmthe results obtained and presented in Examples 1 and 2 above, that thesetumor types show little to no expression of Notch signaling pathwaycomponents, consistent with loss of Notch signaling in NETs or poorlydifferentiated tumors with neuroendocrine phenotypes.

Example 6 Generation of Anti-DLL3 Modulators

DLL3 modulators in the form of murine antibodies were produced inaccordance with the teachings herein through inoculation withrecombinant human DLL3-Fc or with human DLL3-His (each comprising themature ECD of DLL3 set forth in FIG. 1C; SEQ ID NO: 3) in two separateimmunization campaigns. In this regard three strains of mice (Balb/c,CD-1 and FVB) were inoculated with human recombinant DLL3 to providehybridomas secreting high affinity, murine monoclonal antibodymodulators.

The hDLL3-Fc fusion construct was obtained from Adipogen International(Catalog No. AG-40A-0113) where it had been purified from thesupernatant of DLL3-Fc overexpressing HEK 293 cells as described in themanufacturer's product data sheet. Recombinant hDLL3-His protein waspurified from the supernatants of CHOK1 cells engineered to overexpresshDLL3-His. 10 μg of hDLL3-Fc or hDLL3-His immunogen was emulsified withan equal volume of TITERMAX® Gold (CytRx Corporation) or alum adjuvantand used for the immunization of each mouse. The resulting emulsionswere then injected into three female mice (1 each: Balb/c, CD-1 and FVB)via the footpad route.

Solid-phase ELISA assays were used to screen mouse sera for mouse IgGantibodies specific for human DLL3. A positive signal above backgroundwas indicative of antibodies specific for DLL3. Briefly, 96 well plates(VWR International, Cat. #610744) were coated with recombinant DLL3-Hisat 0.5 μg/ml in ELISA coating buffer overnight. After washing with PBScontaining 0.02% (v/v) Tween 20, the wells were blocked with 3% (w/v)BSA in PBS, 200 μl/well for 1 hour at room temperature (RT). Mouse serumwas titrated (1:100, 1:200, 1:400, and 1:800) and added to the DLL3coated plates at 50 μL/well and incubated at RT for 1 hour. The platesare washed and then incubated with 50 μL/well HRP-labeled goatanti-mouse IgG diluted 1:10,000 in 3% BSA-PBS or 2% FCS in PBS for 1hour at RT. Again the plates were washed and 40 μL/well of a TMBsubstrate solution (Thermo Scientific 34028) was added for 15 minutes atRT. After developing, an equal volume of 2N H₂SO₄ was added to stopsubstrate development and the plates were analyzed by spectrophotometerat OD 450.

Sera-positive immunized mice were sacrificed and draining lymph nodes(popliteal and inguinal, and medial iliac if enlarged) were dissectedout and used as a source for antibody producing cells. A single cellsuspension of B cells (228.9×10⁶ cells) was fused with non-secretingP3×63Ag8.653 myeloma cells (ATCC #CRL-1580) at a ratio of 1:1 byelectrofusion. Electrofusion was performed using the BTX Hybrimmune™System, (BTX Harvard Apparatus) as per the manufacturer's directions.After the fusion procedure the cells were resuspended in hybridomaselection medium supplemented with Azaserine (Sigma #A9666), highglucose DMEM medium with sodium pyruvate (Cellgro cat#15-017-CM)containing 15% Fetal Clone I serum (Hyclone), 10% BM Condimed (RocheApplied Sciences), 4 mM L-glutamine, 100 IU Penicillin-Streptomycin and50 μM 2-mercaptoethanol and then plated in three T225 flasks in 90 mLselection medium per flask. The flasks were then placed in a humidified37° C. incubator containing 5% CO₂ and 95% air for 6-7 days.

After six to seven days of growth the library consisting of the cellsgrown in bulk in the T225s was plated at 1 cell per well in Falcon 96well U-bottom plates using the Aria I cell sorter. The selectedhybridomas were then grown in 200 μL of culture medium containing 15%Fetal Clone I serum (Hyclone), 10% BM-Condimed (Roche Applied Sciences),1 mM sodium pyruvate, 4 mM L-glutamine, 100 IU Penicillin-Streptamycin,50 μM 2-mercaptoethanol, and 100 μM hypoxanthine. Any remaining unusedhybridoma library cells were frozen for future library testing. Afterten to eleven days of growth supernatants from each well of the platedcells were assayed for antibodies reactive fur DLL3 by ELISA and FACSassays.

For screening by ELISA 96 well plates were coated with denatured humanDLL3 or cell lysates of 293 cells overexpressing human DLL3 (obtained asdiscussed below), in sodium carbonate buffer overnight at 4° C. Theplates were washed and blocked with 3% BSA in PBS/Tween for one hour at37° C. and used immediately or kept at 4° C. Undiluted hybridomasupernatants were incubated on the plates for one hour at RT. The plateswere washed and probed with HRP labeled goat anti-mouse IgG diluted1:10,000 in 3% BSA-PBS for one hour at RT. The plates were thenincubated with substrate solution as described above and read at OD 450.Wells containing immunoglobulin that preferentially bound human DLL3, asdetermined by a signal above background, were transferred and expanded.

Growth positive hybridoma wells secreting murine immunoglobulin werealso screened for human DLL3 specificity and cynomolgus, rat and murineDLL3 cross reactivity using a flow cytometry based assay with 293 cellsengineered to over-express either human DLL3 (h293-hDLL3), cynomolgusDLL3 (h293-cDLL3), rat (h293-rDLL3) or murine DLL3 (h293-mDLL3)proteins. h293-hDLL3 cells were made by transduction of 293T cells usinga lentivirus made from a commercial bicistronic lentiviral vector (OpenBiosystems) that expressed both hDLL3 and a GFP marker. h293-mDLL3 cellswere made by transduction of 293T cells using a bicistronic lentiviralvector expressing both mDLL3 and a RFP marker, constructed as follows. ADNA fragment (FIG. 10A; SEQ ID NO: 5) encoding the mature murine DLL3protein (FIG. 10B; SEQ ID NO: 6) was obtained by PCR amplification froma commercial murine DLL3 construct (Origene) and subcloned downstream ofan IgG K signal peptide sequence previously engineered upstream of themultiple cloning site of pCDH-EF1-MCS-IRES-RFP (System Biosciences)using standard molecular cloning techniques. Similarly, h293-rDLL3 cellswere made by transduction of 293T cells using a bicistronic lentiviralvector expressing both rat DLL3 and a GFP marker, constructed by cloninga synthetic DNA fragment (GeneWiz) comprising a codon-optimized sequenceencoding the mature rat DLL3 protein (accession NP_144611.8.1, residues25-589) downstream of an IgK signal peptide sequence previouslyengineered upstream of the multiple cloning site ofpCDH-EF1-MCS-IRES-GFP (System Biosciences) using standard molecularcloning techniques. Finally, cynomolgus (e.g., Macaca fascicularis) DLL3(cDLL3) sequence was deduced using the human DLL3 sequence to BLASTagainst the publically available Macaca fascicularis whole-genomeshotgun contigs, and assembling the exon sequences of the Cynomolgusgene assuming maintenance of exonic structure in the gene acrossspecies. PCR amplification and direct sequencing of the individual exons2-7 from Cynomolgus genomic DNA (Zyagen) was used to confirm that thededuced sequence was correct across the ECD region of the protein. ThecDLL3 DNA sequence (FIG. 10C; SEQ ID NO: 7), encoding the cDLL3 protein(FIG. 10D; SEQ ID NO: 8), was manufactured synthetically (GeneWiz) andsubcloned downstream of an IgG K signal peptide sequence previouslyengineered upstream of the multiple cloning site ofpCDH-EF1-MCS-IRES-GFP (System Biosciences) using standard molecularcloning techniques. Transduction of 293T cells with this vector yieldedthe h293-cDLL3 cells.

For the flow cytometry assays, 50×10⁴ h293 cells transduced respectivelywith human, cynomolgus, rat or murine DLL3 were incubated for 30 minuteswith 25-100 μL hybridoma supernatant. Cells were washed with PBS, 2%FCS, twice and then incubated with 50 μL of a goat-anti-mouse IgG Fcfragment specific secondary conjugated to DyLight 649 diluted 1:200 inPBS/2% FCS. After 15 minutes of incubation, cells were washed twice withPBS/2% FCS and re-suspended in PBS/2% FCS with DAPI and analyzed by flowcytometry using a FACSCanto II as per the manufacturer's instructions.Wells containing immunoglobulin that preferentially bound the DLL3⁺ GFP⁺cells were transferred and expanded. The resulting hDLL3 specific clonalhybridomas were cryopreserved in CS-10 freezing medium (BiolifeSolutions) and stored in liquid nitrogen. Antibodies that boundh293-hDLL3, h293-cDLL3, h293-rDLL3 and/or h293-mDLL3 cells were noted ascross-reactive (see FIG. 12). Based on this assay all the selectedmodulators that were cross reactive with the murine antigen were alsocross reactive with the rat antigen.

ELISA and flow cytometry analysis confirmed that purified antibody frommost or all of these hybridomas bound DLL3 in a concentration-dependentmanner. One fusion of each immunization campaign was performed andseeded in 64 plates (6144 wells at approximately 60-70% cloningefficiency). The hDLL3-Fc immunization campaign and screening yieldedapproximately 90 murine antibodies specific for human DLL3, several ofwhich were cross reactive with murine DLL3. The hDLL3-His immunizationcampaign yielded 50 additional murine antibodies specific for humanDLL3, a number of which cross reacted with murine DLL3.

Example 7 Sequencing of Murine DLL3 Modulators

Based on the foregoing, a number of exemplary distinct monoclonalantibodies that bind immobilized human DLL3 or h293-hDLL3 cells withapparently high affinity were selected for sequencing and furtheranalysis. As shown in a tabular fashion in FIGS. 11A and 11B, sequenceanalysis of the light chain variable regions (FIG. 11A) and heavy chainvariable regions (FIG. 11B) from selected monoclonal antibodiesgenerated in Example 6 confirmed that many had novel complementaritydetermining regions and often displayed novel VDJ arrangements. Notethat the complementarity determining regions set forth in FIGS. 11A and11B are defined as per Chothia et al., supra.

As a first step in sequencing exemplary modulators, the selectedhybridoma cells were lysed in Trizol® reagent (Trizol Plus RNAPurification System, Life Technologies) to prepare the RNA. In thisregard between 10⁴ and 10⁵ cells were resuspended in 1 mL Trizol andshaken vigorously after addition of 200 μL of chloroform. Samples werethen centrifuged at 4° C. for 10 minutes and the aqueous phase wastransferred to a fresh microfuge tube where an equal volume ofisopropanol was added. The tubes were again shaken vigorously andallowed to incubate at RT for 10 minutes before being centrifuged at 4°C. for 10 minutes. The resulting RNA pellets were washed once with 1 mLof 70% ethanol and dried briefly at RT before being resuspended in 40 μLof DEPC-treated water. The quality of the RNA preparations wasdetermined by fractionating 3 μL in a 1% agarose gel before being storedat −80° C. until used.

The variable region of the Ig heavy chain of each hybridoma wasamplified using a 5′ primer mix comprising thirty-two mouse specificleader sequence primers, designed to target the complete mouse V_(H)repertoire, in combination with a 3′ mouse Cγ primer specific for allmouse Ig isotypes. A 400 bp PCR fragment of the V_(H) was sequenced fromboth ends using the same PCR primers. Similarly a mix of thirty-two 5′Vκ leader sequence primers designed to amplify each of the Vκ mousefamilies combined with a single reverse primer specific to the mousekappa constant region were used to amplify and sequence the kappa lightchain. The V_(H) and V_(L) transcripts were amplified from 100 ng totalRNA using reverse transcriptase polymerase chain reaction (RT-PCR).

A total of eight RT-PCR reactions were run for each hybridoma: four forthe Vκ light chain and four for the V gamma heavy chain (γ1). The OneStep RT-PCR kit was used for amplification (Qiagen). This kit provides ablend of Sensiscript and Omniscript Reverse Transcriptases, HotStarTaqDNA Polymerase, dNTP mix, buffer and Q-Solution, a novel additive thatenables efficient amplification of “difficult” (e.g., GC-rich)templates. Reaction mixtures were prepared that included 3 μL of RNA,0.5 of 100 μM of 100 μM of either heavy chain or kappa light chainprimers (custom synthesized by IDT), 5 μL of 5× RT-PCR buffer, 1 μLdNTPs, 1 μL of enzyme mix containing reverse transcriptase and DNApolymerase, and 0.4 μL of ribonuclease inhibitor RNasin (1 unit). Thereaction mixture contains all of the reagents required for both reversetranscription and PCR. The thermal cycler program was set for an RT step50° C. for 30 minutes, 95° C. for 15 minutes, followed by 30 cycles ofPCR (95° C. for 30 seconds, 48° C. for 30 seconds, 72° C. for oneminute). There was then a final incubation at 72° C. for 10 minutes.

To prepare the PCR products for direct DNA sequencing, they werepurified using the QIAquick™ PCR Purification Kit (Qiagen) according tothe manufacturer's protocol. The DNA was eluted from the spin columnusing 50 μL of sterile water and then sequenced directly from bothstrands. The extracted PCR products were directly sequenced usingspecific V region primers. Nucleotide sequences were analyzed using IMGTto identify germline V, D and J gene members with the highest sequencehomology. The derived sequences were compared to known germline DNAsequences of the Ig V- and J-regions using V-BASE2 (Retter et al.,supra) and by alignment of V_(H) and V_(L) genes to the mouse germlinedatabase to provide the annotated sequences set forth in FIGS. 11A and11B.

More specifically, FIG. 11A depicts the contiguous amino acid sequencesof ninety-two novel murine light chain variable regions from anti-DLL3antibodies (SEQ ID NOS: 20-202, even numbers) and five humanized lightchain variable regions (SEQ ID NOS: 204-212, even numbers) derived fromrepresentative murine light chains. Similarly, FIG. 11B depicts thecontiguous amino acid sequences of ninety-two novel murine heavy chainvariable regions (SEQ ID NOS: 21-203, odd numbers) from the sameanti-DLL3 antibodies and five humanized heavy chain variable regions(SEQ ID NOS: 205-213, odd numbers) from the same murine antibodiesproviding the humanized light chains. Thus, taken together FIGS. 11A and11B provide the annotated sequences of ninety-two operable murineanti-DLL3 antibodies (termed SC16.3, SC16.4, SC16.5, SC16.7, SC16.8,SC16.10, SC16.11, SC16.13, SC16.15, SC16.18, SC16.19, SC16.20, SC16.21,SC16.22, SC16.23, SC16.25, SC16.26, SC16.29, SC16.30, SC16.31, SC16.34,SC16.35, SC16.36, SC16.38, SC16.41, SC16.42, SC16.45, SC16.47, SC16.49,SC16.50, SC16.52, SC16.55, SC16.56, SC16.57, SC16.58, SC16.61, SC16.62,SC16.63, SC16.65, SC16.67, SC16.68, SC16.72, SC16.73, SC16.78, SC16.79,SC16.80, SC16.81, SC16.84, SC16.88, SC16.101, SC16.103, SC16.104,SC16.105, SC16.106, SC16.107, SC16.108, SC16.109, SC16.110, SC16.111,SC16.113, SC16.114, SC16.115, SC16.116, SC16.117, SC16.118, SC16.120,SC16.121, SC16.122, SC16.123, SC16.124, SC16.125, SC16.126, SC16.129,SC16.130, SC16.131, SC16.132, SC16.133, SC16.134, SC16.135, SC16.136,SC16.137, SC16.138, SC16.139, SC16.140, SC16.141, SC16.142, SC16.143,SC16.144, SC16.147, SC16.148, SC16.149 and SC16.150) and five humanizedantibodies (termed hSC16.13, hSC16.15, hSC16.25, hSC16.34 and hSC16.56).Note that these same designations may refer to the clone that producesthe subject antibody and, as such, the use of any particular designationshould be interpreted in the context of the surrounding disclosure.

For the purposes of the instant application the SEQ ID NOS of eachparticular antibody are sequential. Thus mAb SC16.3 comprises SEQ IDNOS: 20 and 21 for the light and heavy chain variable regionsrespectively. In this regard SC16.4 comprises SEQ ID NOS: 22 and 23,SC16.5 comprises SEQ ID NOS: 24 and 25, and so on. Moreover,corresponding nucleic acid sequences for each antibody amino acidsequence in FIGS. 11A and 11B are appended to the instant application inthe sequence listing filed herewith. In the subject sequence listing theincluded nucleic acid sequences comprise SEQ ID NOS that are two hundredgreater than the corresponding amino acid sequence (light or heavychain). Thus, nucleic acid sequences encoding the light and heavy chainvariable region amino acid sequences of mAb SC16.3 (i.e., SEQ ID NOS: 20and 21) comprise SEQ ID NOS: 220 and 221 in the sequence listing. Inthis regard nucleic acid sequences encoding all of the disclosed lightand heavy chain variable region amino acid sequences, including thoseencoding the humanized constructs, are numbered similarly and compriseSEQ ID NOS: 220-413.

Example 8 Humanization of DLL3 Modulators

As alluded to above, five of the murine antibodies from Example 7 werehumanized using complementarity determining region (CDR) grafting. Humanframeworks for heavy and light chains were selected based on sequenceand structure similarity with respect to functional human germlinegenes. In this regard structural similarity was evaluated by comparingthe mouse canonical CDR structure to human candidates with the samecanonical structures as described in Chothia et al. (supra).

More particularly murine antibodies SC16.13, SC16.15, SC16.25, SC16.34and SC16.56 were humanized using a computer-aided CDR-grafting method(Abysis Database, UCL Business Plc.) and standard molecular engineeringtechniques to provide hSC16.13, hSC16.15, hSC16.25, hSC16.34 andhSC16.56 modulators. The human framework regions of the variable regionswere selected based on their highest sequence homology to the subjectmouse framework sequence and its canonical structure. For the purposesof the humanization analysis the assignment of amino acids to each ofthe CDR domains is in accordance with Kabat et al. numbering (supra).

Molecular engineering procedures were conducted using art-recognizedtechniques. To that end total mRNA was extracted from the hybridomas andamplified as set forth in Example 7 immediately above.

From the nucleotide sequence information, data regarding V, D and J genesegments of the heavy and light chains of subject murine antibodies wereobtained. Based on the sequence data new primer sets specific to theleader sequence of the Ig V_(H) and V_(K) light chain of the antibodieswere designed for cloning of the recombinant monoclonal antibody.Subsequently the V-(D)-J sequences were aligned with mouse Ig germ linesequences. The resulting genetic arrangements for each of the fivehumanized constructs are shown in Table 1 immediately below.

TABLE 1 mAb human VH human DH human JH FW changes human VK human JK FWchanges hSC16.13 IGHV2-5 IGHD1-1 JH6 None IGKV-O2 JK1 None hSC16.15VH1-46 IGHD2-2 JH4 None IGKV-L4 JK4 87F hSC16.25 IGHV2-5 IGHD3-16 JH6None IGVK-A10 JK2 None hSC16.34 IGHV1-3 IGHD3-22 JH4 None IGVK-A20 JK187F hSC16.56 IGHV1-18 IGHD2-21 JH4 None IGKV-L2 JK2 None

The sequences depicted in TABLE 1 correspond to the annotated heavy andlight chain sequences set forth in FIGS. 11A and 11B for the subjectclones. More specifically, the entries in Table 1 above correspond tothe contiguous variable region sequences set forth SEQ ID NOS: 204 and205 (hSC16.13), SEQ ID NOS: 206 and 207 (hSC16.15), SEQ ID NOS: 208 and209 (hSC16.25), SEQ ID NOS: 210 and 211 (hSC16.34) and SEQ ID NOS: 212and 213 (hSC16.56). Furthermore, TABLE 1 shows that very few frameworkchanges were necessary to maintain the favorable properties of thebinding modulators. In this respect there were no framework changes orback mutations made in the heavy chain variable regions and only twoframework modifications were undertaken in the light chain variableregions (i.e., 87F in hSC16.15 and hSC16.34).

Following humanization of all selected antibodies by CDR grafting, theresulting light and heavy chain variable region amino acid sequenceswere analyzed to determine their homology with regard to the murinedonor and human acceptor light and heavy chain variable regions. Theresults, shown in Table 2 immediately below, reveal that the humanizedconstructs consistently exhibited a higher homology with respect to thehuman acceptor sequences than with the murine donor sequences. Moreparticularly, the murine heavy and light chain variable regions show asimilar overall percentage homology to a closest match of human germlinegenes (85%-93%) compared with the homology of the humanized antibodiesand the donor hybridoma protein sequences (74%-83%).

TABLE 2 Homology Homology to to Human Murine Parent mAb (CDR acceptor)(CDR donor) hSC16.13 HC 93% 81% hSC16.13 LC 87% 77% hSC16.15 HC 85% 83%hSC16.15 LC 85% 83% hSC16.25 HC 91% 83% hSC16.25 LC 85% 79% hSC16.34 HC87% 79% hSC16.34 LC 85% 81% hSC16.56 HC 87% 74% hSC16.56 LC 87% 76%

Upon testing, and as will be discussed in more detail below, each of thehumanized constructs exhibited favorable binding characteristics roughlycomparable to those shown by the murine parent antibodies.

Whether humanized or murine, once the nucleic acid sequences of thevariable regions are determined the antibodies of the instant inventionmay be expressed and isolated using art recognized techniques. To thatend synthetic DNA fragments of the chosen heavy chain (humanized ormurine) variable region were cloned into a human IgG1 expression vector.Similarly the variable region light chain DNA fragment (again humanizedor murine) was cloned into a human light chain expression vector. Theselected antibody was then expressed by co-transfection of the derivedheavy and the light chain nucleic acid constructs into CHO cells.

More particularly, one compatible method of antibody productioncomprised directional cloning of murine or humanized variable regiongenes (amplified using PCR) into selected human immunoglobulinexpression vectors. All primers used in Ig gene-specific PCRs includedrestriction sites which allowed direct cloning into expression vectorscontaining human IgG1 heavy chain and light chain constant regions. Inbrief, PCR products were purified with Qiaquick PCR purification kit(Qiagen) followed by digestion with AgeI and XhoI (for the heavy chain)and XmaI and DraIII (for the light chain), respectively. Digested PCRproducts were purified prior to ligation into expression vectors.Ligation reactions were performed in a total volume of 10 μL with 200UT4-DNA Ligase (New England Biolabs), 7.5 μL of digested and purifiedgene-specific PCR product and 25 ng linearized vector DNA. Competent E.coli DH10B bacteria (Life Technologies) were transformed via heat shockat 42° C. with 3 μL ligation product and plated onto ampicillin plates(100 μg/mL). The AgeI-EcoRI fragment of the V_(H) region was thaninserted into the same sites of pEE6.4HuIgG1 expression vector while thesynthetic XmaI-DraIII VK insert was cloned into the XmaI-DraIII sites ofthe respective pEE12.4Hu-Kappa expression vector.

Cells producing the selected antibody were generated by transfection ofHEK 293 cells with the appropriate plasmids using 293 fectin. In thisrespect plasmid DNA was purified with QIAprep Spin columns (Qiagen).Human embryonic kidney (HEK) 293T (ATCC No CRL-11268) cells werecultured in 150 mm plates (Falcon, Becton Dickinson) under standardconditions in Dulbecco's Modified Eagle's Medium (DMEM) supplementedwith 10% heat inactivated FCS, 100 μg/mL streptomycin, 100 U/mLpenicillin G (all from Life Technologies).

For transient transfections cells were grown to 80% confluency. Equalamounts of IgH and corresponding IgL chain vector DNA (12.5 μg of each)was added to 1.5 mL Opti-MEM mixed with 50 μL HEK 293 transfectionreagent in 1.5 mL opti-MEM. The mix was incubated for 30 min at roomtemperature and distributed evenly to the culture plate. Supernatantswere harvested three days after transfeetion, replaced by 20 mL of freshDMEM supplemented with 10% FBS and harvested again at day 6 aftertransfection. Culture supernatants were cleared of cell debris bycentrifugation at 800×g for 10 min and stored at 4° C. Recombinantchimeric and humanized antibodies were purified with Protein C beads (GEHealthcare) and stored under appropriate conditions.

Example 9 Characteristics of DLL3 Modulators

Various methods were used to analyze the binding and immunochemicalcharacteristics of selected DLL3 modulators generated as set forthabove. Specifically, a number of the antibody modulators werecharacterized as to affinity, kinetics, binning, binding location andcross reactivity with regard to human, cynomolgus, rat and mouse antigenrecognition (i.e., using the cells and constructs from Example 6) byart-recognized methods including flow cytometry. Affinities and kineticconstants k_(on) and k_(off) of the selected modulators were measuredusing bio-layer interferometry analysis on a ForteBio RED (ForteBio,Inc.) or surface plasmon resonance using a Biacore 2000 each accordingto the manufacturer's instructions.

The characterization results are set forth in tabular form in FIG. 12where it may be seen that the selected modulators generally exhibitedrelatively high affinities in the nanomolar range and, in many cases,were cross-reactive. FIG. 12 further lists the empirically determinedmodulator bin as well as the DLL3 domain bound by the subject modulatoras determined using yeast mediated antigen fragment expression such asdescribed in more detail in Example 10 immediately below. Additionally,FIG. 12 further includes the ability of the modulators to mediatecytotoxic induced cell killing of an NTX kidney tumor line (% LiveCells) determined as set forth in Example 12 below. Taken together,these data demonstrate the varied binding properties of the disclosedmodulators as well as their potential for use in a pharmaceuticalsetting.

As to antibody binning, a ForteBio RED was used per manufacturer'sinstructions to identify competing antibodies that bound to the same ordifferent bins. Briefly, a reference antibody (Ab1) was captured onto ananti-mouse capture chip, a high concentration of non-binding antibodywas then used to block the chip and a baseline was collected. Monomeric,recombinant human DLL3-Flag (Adipogen International) was then capturedby the specific antibody (Ab1) and the tip was dipped into a well witheither the same antibody (Ab1) as a control or into a well with adifferent test antibody (Ab2). If additional binding was observed with anew antibody, then Ab1 and Ab2 were determined to be in a different bin.If no further binding occurred, as determined by comparing bindinglevels with the control Ab1, then Ab2 was determined to be in the samebin. As known in the art this process can be expanded to screen largelibraries of unique antibodies using a full row of antibodiesrepresenting unique bins in a 96-well plate. In the instant case thisbinning process showed the screened antibodies bound to at least ninedifferent bins (designated as Bins A though I in FIG. 12) on the DLL3protein. Based on the apparent size of the DLL3 antigen (where the ECDis approximately 56 kD) and the resolution of the binning methodologyemployed, it is believed that the nine identified bins represent themajority of the bins present on the DLL3 extracellular antigen.

In addition to evaluating the exemplary modulators as set forth above,flow cytometry was performed in order to confirm that selected SC16antibody modulators can immunospecifically associate with human DLL3 andto determine whether the same modulators cross-react with cynomolgus,rat and/or murine DLL3. More particularly the exemplary murinemodulators were analyzed by flow cytornetry using a FACSCanto II and 293cells over-expressing murine, rat, cynomolgus or human DLL3 (i.e.,h293-hDLL3, h293-cDLL3, h293-rDLL3 and h293-mDLL3 expressing GFP)substantially as described in Example 6 above. In some cases, exemplarymurine modulators were analyzed by flow cytometry using a FACSCanto IIand yeast cells displaying cynomologus DLL3 using the methods describedby Cochran et al. (J Immunol Methods, 287 (1-2):147-158 (2004).

Based on flow cytometry all of the selected antibody modulators werefound to bind to human DLL3 over-expressed on 293 cells (data not shown)while a number of the tested antibodies were found to cross-react withcynomolgus and/or marine DLL3 (all antibodies reacting with mouse alsoreacted with rat). In this regard, and as listed in FIG. 12, it wasfound that eight out of the thirteen modulators that immunospecificallyreact with human DLL3 also react with murine (or rat) DLL3. SpecificallymAbs SC16.4, SC16.8, SC16.15, SC16.34, SC16.39, SC16.46, SC16.51 andSC16.56 were found to cross-react with murine DLL3 to a greater orlesser extent while mAbs SC16.7, SC16.10, SC16.13, SC16.25 and SC16.65did not appreciably associate with murine DLL3. Such results are notunexpected given that murine DLL3 is approximately 83% homologous withisoform 2 of human DLL3 (see FIG. 2B). It will be appreciated that thiscross-reactivity may be advantageously exploited in the context of theinstant invention through the use of animal models in drug discovery anddevelopment.

Besides the aforementioned assays, humanized constructs hSC16.13,hSC16.15, hSC16.25, hSC16.34 and hSC16.56 from Example 8 were analyzedto determine if the CDR grafting process had appreciably altered theirbinding characteristics. In this respect the humanized constructs (CDRgrafted) were compared with “traditional” chimeric antibodies comprisingthe murine parent (or donor) heavy and light chain variable domains anda human constant region substantially equivalent to that used in thehumanized constructs. With these constructs surface plasmon resonance(SPR) was conducted using a Biacore 2000 (GE Healthcare) to identify anysubtle changes in rate constants brought about by the humanizationprocess.

Exemplary results for one of the tested modulators (SC16.15) and atabular summary of the results for each of the humanized and chimericconstructs are shown in FIGS. 13A-13C. Based on a concentration seriesof 25 and 12.5 nM of human DLL3 antigen (generating the curves from topto bottom in the FIGS. 13A and 13B for SC16.15) and using a 1:1 Langmuirbinding model, the K_(D) of the SC16.15 antibody binding to human DLL3antigen was estimated to be 0.2 nM. Similar experiments were then runwith the other humanized constructs and chimeric constructs (data notshown) to provide the affinity values set forth in FIG. 13C. Suchresults indicated that the humanization process had not materiallyimpacted the affinity of the modulators.

Example 10 Domain and Epitope Mapping of DLL3 Modulators

In order to characterize and position the epitopes that the disclosedDLL3 antibody modulators associate with or bind to, domain-level epitopemapping was performed using a modification of the protocol described byCochran et al., 2004 (supra). Briefly, individual domains of DLL3comprising specific amino acid sequences were expressed on the surfaceof yeast, and binding by each DLL3 antibody was determined through flowcytometry.

More specifically, yeast display plasmid constructs were created for theexpression of the following constructs: DLL3 extracellular domain (aminoacids 27-466); DLL1-DLL3 chimera, which consists of the N-terminalregion and DSL domain of DLL1 (amino acids 22-225) fused to EGF-likedomains 1 through 6 of DLL3 (amino acids 220-466); DLL3-DLL1 chimera,which consists of the N-terminal region and DSL domain of DLL3 (aminoacids 27-214) fused to EGF-like domains 1 through 8 of DLL1 (amino acids222-518); EGF-like domain #1 (amino acids 215-249); EGF-like domain #2(amino acids 274-310); EGF-like domains #1 and #2 (amino acids 215-310);EGF-like domain #3 (amino acids 312-351); EGF-like domain #4 (aminoacids 353-389); EGF-like domain #5 (amino acids 391-427); and EGF-likedomain #46 (amino acids 429-465). (For domain information see generallyUniProtKB/Swiss-Prot database entry Q9NYJ7 which is incorporated hereinby reference. Note that the amino acid numbering is by reference to anunprocessed DLL3 protein with a leader sequence such as set forth in SEQID NO. 3.) For analysis of the N-terminal region or the EGF domains as awhole, chimeras with the family member DLL1 (DLL1-DLL3 and DLL3-DLL1)were used as opposed to fragments to minimize potential problems withprotein folding. Domain-mapped antibodies had previously been shown notto cross react with DLL1 indicating that any binding to these constructswas occurring through association with the DLL3 portion of theconstruct. These plasmids were transformed into yeast, which were thengrown and induced as described in Cochran et al.

To test for binding to a particular construct, 200,000 induced yeastcells expressing the desired construct were washed twice in PBS+1 mg/mLBSA (PBSA), and incubated in 50 μL of PBSA with biotinylated anti-HAclone 3F10 (Roche Diagnostics) at 0.1 μg/mL and either 50 nM purifiedantibody or 1:2 dilution of unpurified supernatant from hybridomascultured for 7 days. Cells were incubated for 90 minutes on ice,followed by 2 washes in PBSA. Cells were then incubated in 50 μL PBSAwith the appropriate secondary antibodies: for murine antibodies, Alexa488 conjugated streptavidin, and Alexa 647 conjugated goat anti mouse(both Life Technologies) were added at 1 μg/mL each, and for humanizedor chimeric antibodies, Alexa 647 conjugated streptavidin (LifeTechnologies) and R-phycoerythrin conjugated goat anti human (JacksonImmunoresearch) were added at 1 μg/mL each. After a twenty minuteincubation on ice, cells were washed twice with PBSA and analyzed on aFACS Canto II. Antibodies that bound to DLL3-DLL1 chimera weredesignated as binding to the N-terminal region+DSL. Antibodies thatbound specifically to an epitope present on a particular EGF-like domainwere designated as binding to its respective domain (FIG. 14A).

In order to classify an epitope as conformational (e.g., discontinuous)or linear, yeast displaying the DLL3 extracellular domain was heattreated for 30 minutes at 80° C., then washed twice in ice-cold PBSA.Yeast displaying denatured antigen (denatured yeast) were then subjectedto the same staining protocol and flow cytometry analysis as describedabove. Antibodies that bound to both the denatured and native yeast wereclassified as binding to a linear epitope, whereas antibodies that boundnative yeast but not denatured yeast were classified as conformationallyspecific.

A schematic summary of the domain-level epitope mapping data of theantibodies tested is presented in FIG. 14A, with antibodies binding alinear epitope underlined and, where determined, the corresponding binnoted in parenthesis. A review of FIG. 14A shows that the majority ofmodulators tended to map to epitopes found either in the N-terminal/DSLregion of DLL3 or to the second EGF-like domain. As previously alludedto, FIG. 12 presents similar data regarding bin determination and domainmapping for a number of selected modulators in a tabular form.

To document the ability of the disclosed modulators to effectivelyeliminate tumorigenic cells despite binding to different DLL3 regions,killing data was correlated with domain binding. More particularly, FIG.14B shows modulator mediated in vitro killing of the KDY66 PDX line(derived as set forth in Example 12 below) plotted against the bindingdomain of the selected modulator. These data show that domain specificmodulator killing is somewhat variable as measured using this in vitrokilling assay. However, for modulators that are effective, aninteresting trend appears where maximum killing in each domain increasesas the epitope moves towards the N-terminus in the primary sequence. Inparticular, maximum killing efficiency improves from EGF6 to EGF2, andplateaus across the N-terminal domain, EGF1, and EGF2. Additionally, outof the antibodies tested in this assay, the highest percentage ofefficacious antibodies bind at the N-terminal domain. This suggests thatmodulators that associate or bind with the DSL domain or N-terminalregion of DLL3 may prove to be particularly effective as drugs or astargeting moieties for cytotoxic agents.

Fine epitope mapping was further performed on selected antibodies usingone of two methods. The first method employed the Ph.D.-12 phage displaypeptide library kit (New England Biolabs E8110S) which was used inaccordance with the manufacturer's instructions. Briefly, the antibodyfor epitope mapping was coated overnight at 50 μg/mL in 3 mL 0.1 Msodium bicarbonate solution, pH 8, onto a Nunc MaxiSorp tube (Nunc). Thetube was blocked with 3% BSA solution in bicarbonate solution. Then,10¹¹ input phage in PBS+0.1% Tween-20 was allowed to bind, followed byten consecutive washes at 0.1% Tween-20 to wash away non-binding phage.Remaining phage were eluted with 1 mL 0.2 M glycine for 10 minutes atroom temperature with gentle agitation, followed by neutralization with150 μL 1M Tris-HCl pH 9. Eluted phage were amplified and panned againwith 10¹¹ input phage, using 0.5% Tween-20 during the wash steps toincrease selection stringency. DNA from 24 plaques of the eluted phagefrom the second round was isolated using the Qiaprep M13 Spin kit(Qiagen) and sequenced. Binding of clonal phage was confirmed using anELISA assay, where the mapped antibody or a control antibody is coatedonto an ELISA plate, blocked, and exposed to each phage clone. Phagebinding was detected using horseradish peroxidase conjugated anti-M13antibody (GE Healthcare), and the 1-Step Turbo TMB ELISA solution(Pierce). Phage peptide sequences from specifically binding phage werealigned using Vector NTI (Life Technologies) against the antigen ECDpeptide sequence to determine the epitope of binding.

Alternatively, a yeast display method (Chao et al., Nat Protoc. 1(2):755-768, 2007) was used to epitope map select antibodies. Briefly,libraries of DLL3 ECD mutants were generated with error prone PCR usingnucleotide analogues 8-oxo-2′deoxyguanosine-5′-triphosphate and2′-deoxy-p-nucleoside-5′triphosphate (both from TriLink Bio) for atarget mutagenesis rate of one amino acid mutation per clone. These weretransformed into a yeast display format. Using the technique describedabove for domain-level mapping, the library was stained for HA andantibody binding at 50 nM. Using a FACS Aria (BD), clones that exhibiteda loss of binding compared to wild type DLL3 ECD were sorted. Theseclones were re-grown, and subjected to another round of FACS sorting forloss of binding to the target antibody. Using the Zymoprep Yeast PlasmidMiniprep kit (Zymo Research), individual ECD clones were isolated andsequenced. Where necessary, mutations were reformatted as single-mutantECD clones using the Quikchange site directed mutagenesis kit (Agilent).

Individual ECD clones were next screened to determine whether loss ofbinding was due to a mutation in the epitope, or a mutation that causedmisfolding. Mutations that involved cysteine, proline, and stop codonswere automatically discarded due to the high likelihood of a misfoldingmutation. Remaining ECD clones were then screened for binding to anon-competing, conformationally specific antibody. ECD clones that lostbinding to non-competing, conformationally specific antibodies wereconcluded to contain misfolding mutations, whereas ECD clones thatretained equivalent binding as wild type DLL3 ECD were concluded to beproperly folded. Mutations in the ECD clones in the latter group wereconcluded to be in the epitope. The results are listed in TABLE 3immediately below.

TABLE 3 Antibody Clone Epitope SEQ ID NO: SC16.23 Q93, P94, G95, A96,P97 9 SC16.34 G203, R205, P206 10 SC16.56 G203, R205, P206 10

More particularly, a summary of selected antibodies with their derivedepitopes comprising amino acid residues that are involved in antibodybinding are listed in TABLE 3. In this respect antibodies SC16.34 andSC16.56 apparently interact with common amino acid residues which isconsistent with the binning information and domain mapping results shownin FIG. 14A. Moreover, SC16.23 was found to interact with a distinctcontiguous epitope and was found not to bin with SC16.34 or SC16.56.Note that for the purposes of the appended sequence listing SEQ ID NO:10 will comprise a placeholder amino acid at position 204.

Example 11 Flow Cytometry Based Detection of DLL3 on the Surface ofCells and Immunohistochemical Staining of DLL3 in Tumors

To confirm the immunospecific nature of the disclosed modulators,exemplary SC16 antibody modulators were tested using flow cytometry todetermine their ability to selectively recognize engineered 293 celllines expressing DLL3 protein on their surface. In this regard cellsexpressing DLL3 were produced as set forth substantially in Example 6,exposed to selected modulators and examined by flow cytometry asdescribed herein. Isotype-stained and fluorescence minus one (FMO)controls were employed to confirm staining specificity. As demonstratedby the representative data shown in FIG. 15 for the SC16.56 modulator,some of the SC16 antibodies (e.g., SC16.56) gave strong staining of293-hDLL3 cells (FIG. 15B) and 293-mDLL3 cells (FIG. 15C), but not ofnon-DLL3 expressing parental 293 cells (FIG. 15A). These datademonstrate, via flow cytometry, that the disclosed modulatorsimmunospecifically recognize human DLL3, and in the instance of SC16.56,murine DLL3 as well.

To confirm these findings and demonstrate that DLL3 expression could bedetected on human tumor cells, DLL3 protein expression on the surface ofselected NTX tumors was assessed by flow cytometry using severalexemplary SC16 antibodies. In this regard data for one of theseantibodies, SC16.56, and three particular tumors, OV26, KDY66, and LU37,are set forth in FIG. 16. More specifically, NTX tumors were harvested,dissociated, and co-stained with commercially available anti-mouse CD45,anti-mouse H-2Kd, anti-human EpCAM and the above-describedanti-human/mouse DLL3 (SC16.56) antibodies. Similar to the 293-stainingexperiments described above, isotype-stained and fluorescence minus one(FMO) controls were employed to confirm lack of non-specific staining.As seen in FIG. 16, anti-DLL3 staining was higher in a fraction of thehuman NTX tumor cells, as indicated by the fluorescent profile shift tothe right, and by changes in the mean fluorescence intensity (MFI)values, for the ovarian OV26 NTX (FIG. 16A), kidney KDY66 NTX (FIG.16B), and lung LU37 NTX (FIG. 16C) tumor cell lines. SCLC NTX tumorswere also stained in an identical manner and similarly demonstratedpositive expression of DLL3 (data not shown). These data suggest thatDLL3 protein is expressed on the surface of various NTX tumors andtherefore amenable to modulation using anti-DLL3 antibody typemodulators.

To further corroborate the presence of DLL3 protein and localize it inthe tumor architecture, immunohistochemistry (IHC) was performed onhuman patient tumor-derived NTX tumors, normal human tissues and primarySCLC tumors. More specifically IHC was performed on formalin fixedparaffin embedded (FFPE) tissue sections, using an indirect detectionmethod, including a murine monoclonal primary antibody against DLL3(SC16.65), mouse specific biotin conjugated secondary antibodies,avidin/biotin complex coupled with horse-radish peroxidase, tyramidesignal amplification and DAB detection (Nakene PK 1968; 16:557-60). Whenstaining human patient tumor derived NTX tumors, a mouse IgG blockingstep was used to reduce background due to non-specific binding. SC16.65was first validated and confirmed to be appropriate for IHC by showingspecific staining in 293 cells overexpressing DLL3, but not non-DLL3expressing parental 293 cells, and that staining was diminished in cellstreated with DLL3-targeted hairpins designed and validated to knockdownexpression of DLL3 RNA and protein (see Example 14 below, data notshown). IHC on a panel of xenograft NTX tumors showed that DLL3 islocalized both on the membrane and in the cytoplasm of many of SCLC NTXand NET tumors that previously tested positive for DLL3 mRNA (FIG. 16D).Staining intensity was scored from no staining (−) to high expression(+++) with the percent of positive cells also noted. Staining of normalhuman tissues showed no detectable expression of DLL3 (FIG. 16E).Significantly, staining of primary SCLC tumor samples confirmed that36/43 tumors were positive for DLL3 (FIG. 16F). Chromagranin A (CHGA)staining was also performed to confirm that tumors were indeed SCLCtumors. Most tumors that lacked DLL3 also lacked CHGA staining,indicating these sections might not contain tumor tissue or that thetissue was compromised during processing. Two tumors that testedpositive for DLL3 but were negative for CHGA, were both later stage(IIIa) SCLC tumors. This data suggests that DLL3 provides an effectivetherapeutic target as it is not generally expressed in normal humantissues, but is present in the majority of SCLC tumors.

Example 12 DLL3 Modulators Facilitate Delivery of Cytotoxic Agents

To determine whether DLL3 antibody modulators of the instant inventionare able to mediate the delivery of a cytotoxic agent to live cells, anin vitro cell killing assay was performed using randomly selected DLL3antibody modulators.

Specifically 2,500 cells/well of human KDY66, a NET NTX expressingendogenous DLL3, were dissociated into a single cell suspension andplated on BD Primaria™ plates (BD Biosciences) in growth factorsupplemented serum free media as is known in the art, one day before theaddition of antibodies and toxin. Various concentrations of purifiedDLL3 modulators, such as those described in Examples 6 and 7, and afixed concentration of 4 nM anti-Mouse IgG Fab fragment covalentiylinked to saporin toxin (Advanced Targeting Systems, #IT-48) were addedto the cultures for seven days. For killing on 293-hDLL3, 500 cells/wellwere plated in a single cell suspension and plated on BD tissue cultureplates in DMEM with 10% FBS one day before addition of antibodies andtoxin. Two concentrations of various DLL3 modulators and a fixedconcentration of 2 nM anti-Mouse IgG Fab fragment covalently linked tosaporin were added to the cultures for three days. The ability of thesaporin complexes to internalize and kill cells was determined byenumerating viable cell numbers using Cell Titer Glo® (Promega) as permanufacturer's instructions. Raw luminescence counts using culturescontaining cells with the saporin Fab fragment were set as 100%reference values and all other counts calculated accordingly (referredto as “Normalized RLU”). Using this assay it was demonstrated that asubset of DLL3 antibodies tested at 500 and 50 pM killed KDY66 cells, aswell as a subset of antibodies tested at 250 and 25 pM on 293-hDLL3overexpressing cells (FIG. 17A). Isotype controls did not affect cellcounts as shown by the IgG2a, IgG2b, and MOPC bars at the left of thegraph (FIG. 17A).

A subset of DLL3 modulators showing efficient killing in the first assaydescribed above were tested in dilution to determine EC50 values foractivity. Two such representative antibodies, SC16.34 and SC16.15, areshown in FIG. 17B, in which it was determined that SC16.15 showedefficient killing of OV26, an ovarian NET NTX tumor, with a subpicomolarEC50 (e.g., 0.14 pM) relative to the killing profile shown by SC16.34(e.g., 5.7 pM). As saporin kills cells only upon uptake into thecytoplasm where it inactivates ribosomes, this assay also demonstratesthat internalization may occur upon binding of the DLL3-specificantibody to the cell surface, without the need for additionalcrosslinking or dimerization.

Lastly, LU37 was treated with humanized SC16.15 conjugated to ADC1 orwith a humanized IgG1 control ADC1 (conjugated as per Example 13immediately below). Specifically, 2,500 LU37 NTX cells were plated ineach well on BD PrimariaTM plates (BD Biosciences) in growth factorsupplemented serum free media as is known in the art one day before theaddition of the conjugated antibodies. Various concentrations ofhuIgG1-ADC1 or hSC16.15-ADC were added to the cultures for seven days,and the ability of the cytotoxic agents to kill was determined byenumerating cell numbers (as detailed above). Using this assay it wasdemonstrated that hSC16.15-ADC1 efficiently killed LU37. In contrastto >1,000 ng/ml of control ADC needed to kill 50% of LU37, <10 ng/ml ofhSC16.15-ADC1 killed 50% of LU37 (FIG. 17C).

Example 13 Preparation of DLL3 Antibody-Drug Conjugates

Based on the foregoing results with saporin and to further demonstratethe versatility of the instant invention, anti-DLL3 antibody drugconjugates (DLL3-ADCs) were prepared using covalently linked cytotoxicagents. More specifically, DLL3-ADCs were prepared comprising a linkeras described herein, or in the references immediately below, andselected pyrrolobenzodiazepine (PBD) dimers that were covalentlyattached to the disclosed modulators (see, e.g., U.S.P.Ns. 2011/0256157and 2012/0078028 and U.S. Pat. No. 6,214,345 each of which isincorporated herein by reference in its entirety).

PBD drug-linker combinations were synthesized and purified usingart-recognized techniques in view of the cited references. While variousPBD dimers and linkers were employed to fabricate the selecteddrug-linker combinations, each linker unit comprised a terminalmaleimido moiety with a free sulfhydryl. Using these linkers,conjugations were prepared via partial reduction of the mAb with tris(2-carboxyethyl)-phosphine (TCEP) followed by reaction of reduced Cysresidues with the maleimido-linker payload.

More particularly, the selected DLL3 antibody modulator was reduced with1.3 mol TCEP per mol mAb for 2 hr at 37° C. in 25 mM Tris HCl pH 7.5 and5 mM EDTA buffer. The reaction was allowed to cool to 15° C. and thelinker payload in DMSO was added at a ratio of 2.7 mol/mol mAb followedby an additional amount of DMSO to a final concentration of 6% (v/v).The reaction was allowed to proceed for 1 hour. The unreacteddrug-linker was capped by addition of an excess of N-acetyl cysteine.The DLL3-ADC (or SC16-ADC) was then purified by ion exchange columnusing an AKTA Explorer FPLC system (G.E. Healthcare) to removeaggregated high molecular weight antibody, co-solvent and smallmolecules. The eluted ADC was then buffer-exchanged by tangential flowfiltration (TFF) into formulation buffer followed by concentrationadjustment and addition of a detergent. The final ADC was analyzed forprotein concentration (by measuring UV), aggregation (SEC), drug toantibody ratio (DAR) by reverse phase (RP) HPLC, presence ofunconjugated antibody by hydrophobic interaction chromatography (HIC)HPLC, non-proteinaceous materials by RP HPLC and in vitro cytotoxicityusing a DLL3 expressing cell line.

Using the aforementioned procedure, or substantially similarmethodology, a number of ADCs (i.e., M-[L-D]n) comprising various DLL3modulators and PBD dimers were generated and tested in a variety of invivo and in vitro models. For the purposes of these Examples and theinstant disclosures, such ADCs may generally be termed DLL3-ADCs orSC16-ADCs. Discrete ADCs will be named according to the antibody (e.g.,SC16.13) and the specific linker-cytotoxic agent designation ADC1, ADC2,etc. Thus, exemplary modulators compatible with the instant inventionmay comprise SC16.13-ADC1 or SC16.67-ADC2 where ADC1 and ADC2 representindividual PBD dimer cytotoxic agents (and optionally a linker).

Example 14 Specificity of Anti-DLL3 Antibody-Drug Conjugate MediatedToxicity

To demonstrate that toxicity from anti-DLL3 antibody-drug conjugates isspecific to cells expressing endogenous DLL3, experiments were conductedto show that tumor cells known to have endogenous DLL3 expression are nolonger killed by SC16-ADC in vitro when DLL3 expression is suppressed byknocking down expression of DLL3 mRNA and protein using a short-hairpinRNA (shRNA).

KDY66 is a patient-derived xenograft from a papillary renal cellcarcinoma that exhibits neuroendocrine features and expresses DLL3 mRNAand protein (e.g., see FIG. 7 and FIG. 16B). Expression of DLL3 wasreduced in KDY66 cells by transduction with GIPZ Lentiviral HumanDLL3-targeted shRNA (Thermo Fisher Scientific Inc.) containing ananti-DLL3 shRNA. More specifically the lentiviral vector was generatedthrough transfection of 293T cells with a bicistronic lentiviral plasmidexpressing anti-DLL3 shRNA (DLL3HP2) or a control non-silencing shRNA(DLL3NSHP) in the presence of viral packaging plasmids. Resultinglentiviral particles contained in the supernatant were concentrated andharvested by ultracentrifugation. These particles were then used totransduce the KDY66 cell cultures and introduce the shRNA (i.e., DLL3HP2or NSHP) wherein the anti-DLL3 shRNA binds endogenous DLL3 mRNA andtargets it for destruction thereby preventing translation into DLL3protein. Both vector constructs contained an independent GFP expressionmodule for verification of successful transduction and selection oftransduced cells.

Following transduction, expression of DLL3 was evaluated by flowcytometry. Briefly, a sample of disassociated, single cell suspension ofDLL3HP2-transduced cells were labeled with a DLL3 modulator (SC16.34)conjugated to Alexa Fluor 647 (Life Technologies) and analyzed on a FACSCanto II flow cytometer under standard conditions. To demonstrate areduction of DLL3 protein expression on the surface of the DLL3HP2transduced cells, fluorescence intensity was compared with a similarlyprepared sample of KDY66 DLL3NSHP cells stained with a non-reactivecontrol antibody (647-IgG1) and KDY66 DLL3NSHP cells stained with647-DLL3. DLL3NSHP.KDY66 cells were found to exhibit DLL3 proteinexpression substantially equivalent to naïve KDY66 cells (data notshown). As seen in FIG. 18A, DLL3 protein surface expression was reducedin cells transduced with DLL3HP2 compared with naïve cells stained withthe same AlexaFluor-647 labeled antibody.

In order to examine the consequences of DLL3 expression on the growth oftumors DLL3HP2 transduced cells (DLL3⁻) and naïve KDY66 cells (DLL3⁺)were transplanted into immunodeficient mice. From the sample prepared asdescribed above, live human GFP⁺ cells were sorted to collect cells thatcontain the anti-DLL3 shRNA. Five-mouse cohorts were injected (140cells/mouse) with either DLL3HP2 or naïve KDY66 cells and tumor growthwas monitored weekly. From each cohort, two of five recipients grewtumors. Tumor formation in the two DLL3HP2.KDY66 recipients laggedroughly 22 days behind tumor formation in the two naïve KDY66 recipients(FIG. 18B). This observed delay in growth suggests that DLL3 expressionmay be connected to increased or accelerated tumor formation sinceknockdown of DLL3 impacted tumor growth.

As they reached the appropriate volume for randomization (˜160 mm³), theDLL3HP2 KDY66 tumors and naïve KDY66 tumors were harvested fromrecipient mice and dispersed into suspensions of single cells. Continuedreduction of DLL3 expression (i.e., that DLL3 expression was not inducedduring in vivo growth) in DLL3HP2 cells was confirmed on suspensions ofsingle tumor cells by flow cytometry as described above. In this respectFIG. 18C shows that DLL3HP2 transduced cells grown in vitro show reducedexpression of DLL3 protein when compared to naïve cells grown in similarconditions.

Using standard biochemical techniques naïve KDY66 cells or DLL3HP2 KDY66cells were plated into 96 well plates and grown in serum-free media. Adilution series of either humanized hSC16.56-ADC1 (SC16-ADC1) orhumanized anti-hapten IgG-ADC1 (as a control) antibody-drug conjugatesproduced as set forth above were added to cells in triplicate. After 7days of exposure to antibody-drug conjugate, the quantity of live cellswas measured with a luminescence-based detection of ATP in the celllysates of each well (Cell Titer Glo, Promega) substantially as setforth in Example 12.

While 50% of naïve KDY66 cells were killed by a relatively low dose of13.27 pM SC16-ADC, no dose of SC16-ADC1 was able to kill even 20% ofDLL3HP2.KDY66 cells (FIGS. 18D and 18E). Of note, loss of endogenousDLL3 protein expression resulted in a complete loss of in vitro killingby SC16-ADC1. This demonstrates that hSC16-ADC1 cytotoxicity isspecifically targeted to DLL3-expressing cells with little, if any,non-specific toxicity.

Example 15 Conjugated DLL3 Modulators Suppress Tumor Growth

Based on the aforementioned results work was undertaken to demonstratethat conjugated DLL3 modulators of the instant invention shrink andsuppress growth of DLL3 expressing human tumors in vivo. In this regarda number of selected murine antibody modulators were covalentlyassociated with a PBD cytotoxic agent and the resulting ADCs were testedto demonstrate their ability to suppress human NTX tumor growth inimmunodeficient mice.

To this end patient-derived NTX tumors were grown subcutaneously in theflanks of female NOD/SCID recipient mice using art-recognizedtechniques. Tumor volumes and mouse weights were monitored twice weekly.When tumor volumes reached 150-250 mm³, mice were randomly assigned totreatment groups and injected with indicated doses of SC16-ADC2 or ananti-hapten control IgG1-ADC2 (each produced substantially as describedin Example 13 above using the PBD dimer ADC2) via intraperitonealinjection. Mice were given three equal injections, spaced evenly acrossseven days. Following treatment, tumor volumes and mouse weights weremonitored until tumors exceeded 800 mm³ or mice became sick. For alltests, treated mice exhibited no adverse health effects beyond thosetypically seen in immunodeficient tumor-bearing NOD/SCID mice.

FIG. 19 shows the impact of the disclosed ADCs on tumor growth in micebearing different lung tumors exhibiting neuroendocrine features (twosmall cell lung cancer and one large cell lung cancer withneuroendocrine features). In this respect treatment of LU37, a largecell neuroendocrine lung carcinoma, with three exemplary modulators(SC16.13, SC16.46 and SC16.67) conjugated to ADC2 resulted in tumorgrowth suppression lasting as long as 20 days in the case ofSC16.13-ADC2 and SC16.67-ADC2 (FIG. 19A); conversely, though SC16.46moderately reduced tumor growth it exhibited less activity than theother tested modulators. Similarly, treatment of LU73, a small cell lungcarcinoma, with four exemplary modulators (SC16.4, SC16.13, SC16.15 andSC16.46) produced durable remissions lasting, in some cases, beyond 120days post-treatment (FIG. 19B). However, as with the antibodies testedagainst LU37, the antibodies tested against LU73 varied somewhat in theduration of tumor repression. Finally, treatment of LU86, another smallcell lung carcinoma, with two conjugated modulators (SC16.46-ADC2 andSC16.67-ADC2) produced tumor shrinkage with a time to progression of 40days in one case (SC16.67-ADC2; FIG. 19C). Note that in FIG. 19C two ofthe curves substantially overlap (mIgG1-ADC2 and SC16.46-ADC2) and aredifficult to distinguish.

The surprising ability of a variety of conjugated modulators todramatically retard or suppress tumor growth in vivo for extendedperiods further validates the use of the DLL3 as a therapeutic targetfor the treatment of proliferative disorders.

Example 16 Humanized DLL3-ADC Modulators Suppress Tumor Growtb

Given the impressive results provided by DLL3-ADC2, additionalexperiments were performed to demonstrate the efficacy of exemplaryhumanized ADC modulators in treating various types of tumors (includingovarian, lung and kidney cancer) in vivo. Specifically, selectedhumanized anti-DLL3 antibodies (hSC16.13, hSC16.15, hSC16.34 andhSC16.56 produced as set forth in Example 8 above) were conjugated (viaa linker unit) to two discrete PBD cytotoxic agents (ADC1 and ADC2) asdescribed above and, with controls, administered to NTX tumor implantedimmunodeficient mice as set forth in the previous Example. In eachstudy, tumor volumes and mouse weights of the control animals weremonitored until tumors exceeded 800 mm³ or mice became sick. The resultsof these experiments are presented in FIGS. 20A to 20F.

A review of FIGS. 20A-20F show that tumor volume reduction and durableremission was achieved in various tumor types, some exhibitingneuroendocrine features, following treatment with 1 mg/kg hSC16-ADC. Forexample, treatment regimens, where administration is delineated by thevertical lines in the subject FIGS., produced complete and durableeliminations of tumor mass in ovarian carcinoma with neuroendocrinefeatures (OV26, hSC16.15-ADC2, FIG. 20A), a papillary renal cellcarcinoma with neuroendocrine features (KDY66, hSC16.34-ADC1, FIG. 20E)and three small cell lung carcinomas (LU86, hSC16.13-ADC1, FIG. 20B),(LU64, hSC16.13-ADC1, FIG. 20C; LU64, hSC16.13-ADC2+hSC16.13-ADC1, FIG.20D). Absence of tumor recurrence was observed for more than 100 days inall these cases, and in some cases beyond 225 days post-treatment wheremice were followed for an extended period of time. Additionally,treatment with the disclosed modulators produced tumor volume reductionand growth suppression in a clear cell renal cell carcinoma xenograftthat exhibits high levels of DLL3 using a lower dose of 0.5 mg/kg(KDY27, hSC16.56-ADC1, FIG. 20F).

Finally, it should be noted that certain recurrent tumors remainedsensitive to hSC16-ADC toxicity. Eighty days after initial treatmentwith SC16.13-ADC2, recurrence was observed in LU64 (FIG. 20D). Treatmentof recurrent tumors with hSC16.13-ADC1 resulted in elimination ofobservable tumor mass that persisted more than 100 days after the secondtreatment.

Again these results demonstrate the surprising versatility andapplicability of the modulators of the instant invention in treating avariety of proliferative disorders.

Example 17 Reduction of Cancer Stem Cell Frequency by DLL3 Antibody-DrugConjugates

As shown in the previous Examples the disclosed modulators are extremelyeffective in suppressing tumor growth, particularly in ADC form.Moreover, as demonstrated above, DLL3 expression is associated withcancer stem cells that are generally known to be both drug resistant andfuel tumor recurrence and metastasis. Accordingly, to demonstrate thattreatment with DLL3-ADCs reduces the recurrence potential of NTX lines,in vivo limiting dilution assays (LDA) were performed to determine thefrequency of tumor-initiating cells (TIC) in small cell lung cancertumors following treatment with hSC16.13-ADC1 (labeled SC16-ADC in FIG.21).

Patient-derived small cell lung cancer xenograft tumors (LU95 and LU64)were grown subcutaneously in immunodeficient host mice. When tumorvolumes averaged 150 mm³-250 mm³, the mice were randomly segregated intotwo groups of seven mice. Via intraperitoneal injection, mice wereinjected on days 0, 4 and 7 (FIGS. 21A and 21D, dashed vertical lines),with either human IgG1-ADC1 (1 mg/kg; n=7 mice) as a negative control orhSC16.13-ADC1 (1 mg/kg; n=7 mice). On day 8, two representative micefrom each group were euthanized and their tumors were harvested anddispersed to single-cell suspensions. As shown in FIGS. 21A and 21Dwhile tumors treated with hIgG1-ADC1 (IgG1-ADC) continued to grow in thefive remaining mice, volumes of tumors treated with hSC16.13-ADC1(SC16-ADC) were reduced to zero or nearly zero in the five remainingmice.

Using standard flow cytometry techniques and a labeled anti-DLL3antibody, the two harvested tumors from each of the two treatment groupswere confirmed to have similarly positive DLL3 expression. The tumorscells from each respective treatment group were then pooled and livehuman cells were isolated by FACS using a FACSAria III (BectonDickenson) in accordance with the manufacturer's instructions andart-recognized techniques. Briefly, the cells were labeled with FITCconjugated anti-murine H2Kd and anti-murine CD45 antibodies (bothBioLegend, Inc.) and then resuspended in 1 μg/ml DAPI. The resultingsuspension was then sorted under standard conditions with DAPI⁻, mH2Kd⁻and mCD45⁻ human cells being collected and the murine cells beingdiscarded.

Cohorts of five recipient mice were then transplanted with either 2000,500, 120 or 30 sorted live human cells from tumors treated withhSC16.13-ADC1. For comparison, cohorts of five recipient mice weretransplanted with either 1000, 250, 60 or 15 sorted live human cellsfrom tumors treated with the control IgG1-ADC1. Tumors in recipient micewere measured weekly, and individual mice were euthanized before tumorsreached 1500 mm³. After the onset of tumor growth, the study was endedafter four consecutive weeks without a new tumor appearing in anyadditional mouse. At that time, recipient mice were scored as positiveor negative for tumor growth, with positive growth having volumesexceeding 100 mm³.

Across all injected cells doses, recipients of LU95 cells treated withhSC16.13-ADC1 produced only one tumor, compared to twelve in recipientsof LU95 cells treated with IgG1-ADC1 (FIG. 21B). Similarly, recipientsof LU64 cells treated with SC16.13-ADC1 produced three tumors, comparedto 13 tumors in recipients of LU64 cells treated with IgG1-ADC1 (FIG.21E).

Using Poisson distribution statistics (L-Calc software, StemcellTechnologies), injected cell doses of recipients with and without tumorsat 18 weeks post-transplant were used to calculate the frequencies oftumor-initiating cells in each population. The number of TIC per 10,000live human cells in LU95 was reduced more than 100-fold, from 78.1 intumors treated with IgG1-ADC to 0.769 in tumors treated withhSC16.13-ADC1 (FIG. 21C, from 1:128 cells in the control treated to1:12,998 in the modulator treated). In LU64, the number of TIC wasreduced 16.6-fold, from 47.4 TIC to 2.86 TIC per 10,000 live human cellsin tumors treated with IgG1-ADC1 or hSC16.13-ADC1, respectively (FIG.21F, from 1:211 cells in the control treated to 1:3,500 cells in themodulator treated). This substantial reduction in TIC (e.g., cancer stemcell) frequency demonstrates that, in addition to reducing tumor volumesas previously demonstrated, the modulators of the instant invention aresignificantly and specifically reducing cancer stem cell populationsand, by extension, the recurrence, metastatic and re-growth potential ofthe tumors. This reduction in recurrence and re-growth potential arestrongly evidenced by the significant tumor-free survival observed inthe forgoing Examples.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forexample, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PBD,and translations from annotated coding regions in GenBank and RefSeqcited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

The invention claimed is:
 1. A multivalent antibody comprising (a) afirst target binding site that specifically binds to an epitope withinthe DSL domain of a DLL3 protein set forth in SEQ ID NO: 3 or 4, and (b)a second target binding site that binds to an epitope on a differentmolecule.
 2. The multivalent antibody of claim 1, wherein the firsttarget binding site specifically binds to an epitope comprising aminoacids G203, R205, and P206 (SEQ ID NO:10).
 3. The multivalent antibodyof claim 1, wherein the multivalent antibody competes for binding to ahuman DLL3 protein with an antibody comprising a light chain variableregion set forth as SEQ ID NO: 84 and a heavy chain variable region setforth as SEQ ID NO:
 85. 4. The multivalent antibody of claim 1, whereinthe first target binding site comprises three CDRs of a light chainvariable region set forth as SEQ ID NO: 84 and three CDRs of a heavychain variable region set forth as SEQ ID NO:
 85. 5. The multivalentantibody of claim 1, wherein the first target binding site comprisesresidues 24-34 of SEQ ID NO: 84 for CDR-L1, residues 50-56 of SEQ ID NO:84 for CDR-L2, residues 89-97 of SEQ ID NO: 84 for CDR-L3, residues31-35 of SEQ ID NO: 85 for CDR-H1, residues 50-65 of SEQ ID NO: 85 forCDR-H2, and residues 95-102 of SEQ ID NO: 85 for CDR-H3, wherein theresidues are numbered according to Kabat.
 6. The multivalent antibody ofclaim 1, wherein the second target binding site binds to an epitope on acytotoxic T-cell.
 7. The multivalent antibody of claim 6, wherein thesecond target binding site binds to an epitope on a CD3 protein.
 8. Themultivalent antibody of claim 1, wherein the multivalent antibody is abispecific antibody.
 9. The multivalent antibody of claim 8, wherein thebispecific antibody is a single chain antibody.
 10. A multivalentantibody comprising (a) a first target binding site that specificallybinds to an epitope on a DLL3 protein, and (b) a second target bindingsite that binds to an epitope on a cytotoxic T-cell.
 11. The multivalentantibody of claim 10, wherein the first target binding site specificallybinds to an epitope within the DSL domain of a DLL3 protein set forth inSEQ ID NO: 3 or
 4. 12. The multivalent antibody of claim 10, wherein thefirst target binding site specifically binds to an epitope comprisingamino acids G203, R205, and P206 (SEQ ID NO:10).
 13. The multivalentantibody of claim 10, wherein the multivalent antibody competes forbinding to a human DLL3 protein with an antibody comprising a lightchain variable region set forth as SEQ ID NO: 84 and a heavy chainvariable region set forth as SEQ ID NO:
 85. 14. The multivalent antibodyof claim 10, wherein the first target binding site comprises three CDRsof a light chain variable region set forth as SEQ ID NO: 84 and threeCDRs of a heavy chain variable region set forth as SEQ ID NO:
 85. 15.The multivalent antibody of claim 10, wherein the first target bindingsite comprises residues 24-34 of SEQ ID NO: 84 for CDR-L1, residues50-56 of SEQ ID NO: 84 for CDR-L2, residues 89-97 of SEQ ID NO: 84 forCDR-L3, residues 31-35 of SEQ ID NO: 85 for CDR-H1, residues 50-65 ofSEQ ID NO: 85 for CDR-H2, and residues 95-102 of SEQ ID NO: 85 forCDR-H3, wherein the residues are numbered according to Kabat.
 16. Themultivalent antibody of claim 10, wherein the second target binding siteis an epitope on a CD3 protein expressed on a cytotoxic T-cell.
 17. Themultivalent antibody of claim 10, wherein the multivalent antibody is abispecific antibody.
 18. The multivalent antibody of claim 17, whereinthe bispecific antibody is a single chain antibody.
 19. A bispecificantibody comprising (a) a first target binding site that specificallybinds to an epitope on a DLL3 protein, and (b) a second target bindingsite that binds to an epitope on a CD3 protein expressed on a cytotoxicT-cell.
 20. The bispecific antibody of claim 19, wherein the firsttarget binding site specifically binds to an epitope within the DSLdomain of a DLL3 protein set forth in SEQ ID NO: 3 or
 4. 21. Thebispecific antibody of claim 19, wherein the first target binding sitespecifically binds to an epitope comprising amino acids G203, R205, andP206 (SEQ ID NO:10).
 22. The bispecific antibody of claim 19, whereinthe bispecific antibody competes for binding to a human DLL3 proteinwith an antibody comprising a light chain variable region set forth asSEQ ID NO: 84 and a heavy chain variable region set forth as SEQ ID NO:85.
 23. The bispecific antibody of claim 19, wherein the first targetbinding site comprises three CDRs of a light chain variable region setforth as SEQ ID NO: 84 and three CDRs of a heavy chain variable regionset forth as SEQ ID NO:
 85. 24. The bispecific antibody of claim 19,wherein the first target binding site comprises residues 24-34 of SEQ IDNO: 84 for CDR-L1, residues 50-56 of SEQ ID NO: 84 for CDR-L2, residues89-97 of SEQ ID NO: 84 for CDR-L3, residues 31-35 of SEQ ID NO: 85 forCDR-H1, residues 50-65 of SEQ ID NO: 85 for CDR-H2, and residues 95-102of SEQ ID NO: 85 for CDR-H3, wherein the residues are numbered accordingto Kabat.
 25. The bispecific antibody of claim 19, wherein thebispecific antibody is a single chain antibody.
 26. A bispecificantibody comprising (a) a first target binding site that specificallybinds to an epitope on a DLL3 protein, wherein the first target bindingsite comprises three CDRs of a light chain variable region set forth asSEQ ID NO: 84 and three CDRs of a heavy chain variable region set forthas SEQ ID NO: 85; and (b) a second target binding site that binds to anepitope on a CD3 protein.
 27. The bispecific antibody of claim 26,wherein the first target binding site comprises residues 24-34 of SEQ IDNO: 84 for CDR-L1, residues 50-56 of SEQ ID NO: 84 for CDR-L2, residues89-97 of SEQ ID NO: 84 for CDR-L3, residues 31-35 of SEQ ID NO: 85 forCDR-H1, residues 50-65 of SEQ ID NO: 85 for CDR-H2, and residues 95-102of SEQ ID NO: 85 for CDR-H3, wherein the residues are numbered accordingto Kabat.
 28. The bispecific antibody of claim 26, wherein thebispecific antibody is a single chain antibody.